Patent Publication Number: US-2021172017-A1

Title: Methods of in vivo evaluation of gene function

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 17/025,784, filed on Sep. 18, 2020, entitled “METHODS OF IN VIVO EVALUATION OF GENE FUNCTION,” which claims the benefit of and priority to U.S. Provisional Patent Application No. 62/902,932, filed on Sep. 19, 2019 entitled “METHODS OF IN VIVO EVALUATION OF GENE FUNCTION,” the contents of which are incorporated by reference herein in their entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under Grant No. MI-1115727, MI-1096066, MH094271, HG009761, MI-1110049, HL141201, and HG006193 awarded by National Institutes of Health. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled BROD-4900US-CON_ST25.txt, created on Jan. 29, 2021 and having a size of 21,016 bytes (25 KB on disk). The content of the sequence listing is incorporated herein in its entirety. 
     TECHNICAL FIELD 
     The subject matter disclosed herein is generally directed to methods and uses thereof for in vivo evaluation of gene functions. 
     BACKGROUND 
     In the past decades, human genetics has uncovered strong links between genetic states and human diseases ranging from breast cancer to psychiatric disorders (1). Many risk-associated genes for a variety of diseases have been proposed. However, a major challenge remains for the identification of the point of action of these risk-associated genes, because each can affect any of a massive number of different tissues, cell types, and molecular pathways. High-resolution phenotyping methods to identify tissue- and cell-type specific effects of genetic perturbations are needed, as generating and analyzing individual knockout animal models for long lists of risk-associated genes is prohibitive as a first line of functional investigation. 
     Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention. 
     SUMMARY 
     In certain example embodiments, methods are provided for in vivo evaluating a plurality of genes in parallel. The methods comprise introducing a plurality of genetic perturbations into a plurality of progenitor cells in an in vivo animal model. The genetic perturbation is achieved by introducing mutations into each gene of the plurality of genes in vivo. In some embodiments, the genetic perturbation is achieved by introducing mutations into each gene of the plurality of genes in utero. In the meantime, a reporter gene is also introduced together with each genetic perturbation. As the progenitor cells develop into diverse distinct types of progeny cells, the genomic, genetic, epigenetic, proteomic, or phenotypic profiles of distinct types of progeny cells are profiled, thus revealing the function of each gene in the plurality of genes in a cell type-specific manner. 
     In certain embodiments, methods and uses of preparing guide RNAs targeting the gene of interest are provided. Using these methods, one can effectively prepare a plurality of lentivirus-based genetic perturbations targeting a plurality of genes of interest in parallel. 
     In certain embodiments, methods of in utero genetic perturbation are provided. The methods allow one to specifically deliver genetic perturbations into desired progenitor cells in utero. 
     In certain embodiments, methods of preparing desired progeny cells are provided. These methods allow one to effectively obtain single progeny cells for subsequent analyses. 
     In certain embodiments, methods of analyzing perturbed progeny cells are provided. In some embodiments, progeny cells are subjected to single-cell RNA sequencing, wherein a transcriptome for each cell with single or multiplex genetic perturbation can be obtained. 
     In certain embodiments, methods and uses of evaluating gene functions based on the changes in gene expression programs, cell states, and other genomic and proteomic parameters are provided. Using these methods, the in vivo functions of multiple genes can be revealed in parallel in a cell type-specific manner. 
     Described in certain example embodiments herein are methods of identifying functions of a plurality of genes in parallel in vivo, comprising:
         a. introducing, in vivo, a plurality of genetic perturbations in each of a plurality of progenitor cells in a Cas animal model, wherein each genetic perturbation is operatively coupled to a reporter gene and a barcode;   b. generating an enriched perturbed cell population by enriching for cells expressing the reporter;   c. identifying cell types and corresponding perturbations via scRNA-seq in the enriched perturbed cell population; and   d. detecting one or more gene modules that co-vary within a cell type in the enriched perturbed cell population.       

     In certain example embodiments, the enriched perturbed cell population comprises progenitor cell progeny. 
     In certain example embodiments, the plurality of genetic perturbations are introduced using two or more guide RNAs (gRNAs) for each target gene, wherein the two or more gRNAs each bind to s sequence of an exon, an intron, or both at the 5′ end of a target gene. 
     In certain example embodiments, the two or more gRNAs each bind to a sequence of a coding exon at the 5′ end of a target gene. 
     In certain example embodiments, each of the two or more gRNAs are controlled by a different pol III promoter. 
     In certain example embodiments, a first gRNA of the two or more gRNAs is controlled by a human pol III promoter and a second gRNA of the two or more gRNAs is controlled by a non-human pol III promoter. 
     In certain example embodiments, the human pol III promoter and the non-human pol III promoters are each independently selected from a U6, a 7SK, or an H1 promoter. 
     In certain example embodiments, one or more of the poll III promoters are constitutive. 
     In certain example embodiments, one or more of the pol III promoters are inducible. 
     In certain example embodiments, the barcode is polyadenylated. 
     In certain example embodiments, the reporter gene is controlled by a constitutive pol II promoter. 
     In certain example embodiments, introducing further comprises delivering to the plurality of progenitor cells a pool of engineered virus particles comprising equal genetic perturbation representation. 
     In certain example embodiments, the engineered virus particles are engineered lentiviral particles. 
     In certain example embodiments, introducing further comprises delivering the pool of engineered virus particles to a target tissue of a developing embryo of the Cas animal model in utero. 
     In certain example embodiments, the developing embryo is at stage between E5-E15 or an equivalent stage thereof. 
     In certain example embodiments, the reporter gene encodes an optically active protein. 
     In certain example embodiments, the reporter gene encodes a cell surface molecules selected from the group of: CD3, CD4, CD19, CD20, CD22, CD34, CD45, CD80, a cell surface receptor, a cluster differentiation (CD) molecule, or any combination thereof. 
     In certain example embodiments, the Cas animal model constitutively or inducibly expresses a Cas protein in one of, a plurality of, or all of its cells. 
     In certain example embodiments, the Cas protein is a Cas Type I, II, III, IV, or V protein. 
     In certain example embodiments, identifying further comprises a genomic analysis, an epigenomic analysis, a transcriptomic analysis, a proteomic analysis, or a combination thereof. 
     In certain example embodiments, the method further comprises a genomic analysis, an epigenomic analysis, a transcriptomic analysis, a proteomic analysis, or a combination thereof. 
     In certain example embodiments, the plurality of genes are autism spectrum disorder associated genes. 
     Described in certain example embodiments herein are methods of in vivo screening for therapeutic targets useful for developing treatment for a disease, comprising:
         a. performing a method as in any of paragraphs e.g. [0013]-[0034] and as further described elsewhere herein, wherein the plurality of genes are a plurality of candidate genes; and   b. selecting one or more candidate genes that produce a change in one or more identified gene modules that are indicative of the disease status; whereby the selected one or more candidate genes are identified as therapeutic targets for disease treatment screening.       

     In certain example embodiments, the method further comprises using the selected candidate gene(s) as therapeutic targets in a disease treatment screen. 
     In certain example embodiments, the disease treatment screen is an autism spectrum disease treatment screen. 
     In certain example embodiments, the disease is an autism spectrum disease. 
     Described in certain example embodiments herein are therapeutic agents for treating a disease where the therapeutic agent is capable of modifying the function, activity, expression, or a combination thereof of identified therapeutic targets of any one of claims [0035]-[0038] and as further described elsewhere herein, one or more gene product(s) thereof, or both. 
     In certain exemplary embodiments, the disease is an autism spectrum disease. 
     These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which: 
         FIGS. 1A-1E —In vivo Perturb-Seq to investigate functions of a panel of ASD/ND risk genes harboring de novo variants. ( FIG. 1A ) Schematics of the in vivo Perturb-Seq platform, which introduces mutations in individual genes in utero at E12.5, followed by transcriptomic profiling of the cellular progeny of these perturbed cells at P7 via single-cell RNA sequencing (scRNA-seq). ( FIG. 1B ) tSNE of five major cell populations identified in the Perturb-Seq cells. ( FIG. 1C ) In vivo Perturb-Seq lentiviral vector carrying an mCherry reporter drives detectable expression within 24 h, and can sparsely infect brain cells across many brain regions. Scale bar is 1000 ( FIG. 1D ) Cell-type analysis of in vivo Perturb-Seq of ASD/ND de novo risk genes. Canonical marker genes were used to identify major cell clusters (left), and cell-type distribution in each perturbation group (right). Negative control (GFP) is highlighted by a black rectangle. ( FIG. 1E ) tSNEs showing the subclusters of each of the five major cell types, identified by re-clustering each cell type separately. 
         FIGS. 2A-2C —In vivo Perturb-Seq reveals cell-type specific effects of ASD/ND risk gene perturbations. ( FIG. 2A ) Schematic illustration of the Perturb-Seq analysis pipeline. ( FIG. 2B ) ASD/ND risk gene perturbation effects in different WGCNA gene modules compared to GFP controls. Dot color corresponds to effect size, dot size corresponds to negative base 10 log(P-value). Module gene lists are presented in Table 4. P-values were calculated from linear modeling, Padj was calculated by Benjamini &amp; Hochberg FDR correction. ( FIG. 2C ) The four cell types and five gene modules that were altered by ASD risk gene perturbations. Top row: subcluster tSNE of each cell class (repeated from  FIG. 1E  for ease of comparison). Bottom row: feature plots of gene module expression scores and the top correlated genes within each module across the relevant cell class. 
         FIGS. 3A-3F —Perturbation effect in oligodendrocytes and validation in the Chd8+/− mouse model. ( FIG. 3A ) tSNE of oligodendrocyte subtypes from the Perturb-Seq data. ( FIG. 3B ) The ODC1 gene module expression score in each cell (left) and in each subcluster (right). ( FIG. 3C ) Average expression of genes in the ODC1 gene module (by row) in each perturbation group (by column), scaled by row. ( FIG. 3D ) Effect size of each perturbation on the ODC1 gene module compared to the control group. Note that the perturbation effects of the different genes present a continuous gradient. Error bars represent 95% confidence intervals. ( FIG. 3E ) In situ hybridization for Cspg4, a gene in module ODC1 that is a known marker of oligodendrocyte precursor cells (OPC), in the somatosensory cortex of P7 Chd8+/− and wild-type littermates. The bottom images of represent the higher magnifications of top images, and the right images represent higher magnifications for each cell. Right: quantification of Cspg4 expression in P7 cortex of Chd8+/− and wild-type littermates. Each dot represents the gene expression value from one cell; error bars represent standard error of the mean (n=3 animals per genotype). Scale bar is 1000 μm (left bottom panel), 100 μm (left top panel), and 10 μm (right panel), respectively. ( FIG. 3F ) Immunohistochemistry for PDGFRA and MBP (markers for immature OPC and mature oligodendrocytes, respectively), PDGFRA+cell counts, and distribution of MBP expression, in the somatosensory cortex of P11 Chd8+/− animals and wild-type littermates. Scale bar is 1000 μm (left panel) and 250 μm (right panel), respectively. 
         FIGS. 4A-4F —Cell-type specific gene modules from Perturb-Seq are conserved in developing human brains. ( FIG. 4A ) Percent of genes with a human orthologue expressed in &gt;5% of cells of the associated cell type in scRNA-seq datasets from the human brain or human brain organoids. ( FIG. 4B ) Normalized average pairwise correlation of gene expression within each gene module in the human brain or human brain organoids. Correlation values were normalized to the mean correlation from the background distribution, and divided by the standard deviation of the background distribution. Correlations are shown for modules with at least 4 genes after filtering out genes expressed in less than 5% of cells. Bars represent 95% confidence intervals. Red color represents statistical significance (FDR&lt;0.05). ( FIG. 4C ) Expression of module PN3 over developmental time in human brain tissues across regions (BrainSpan data). ( FIG. 4D ) Expression of each module over developmental time in human primary somatosensory cortex S1C (BrainSpan). ( FIG. 4E ) Distribution of the Spearman correlation of module expression with age in human brain data over various brain regions (BrainSpan). ( FIG. 4F ) Differential gene expression analysis of human prefrontal cortical samples from ASD donors and controls. Left: Expression of differentially expressed (DE) genes across cell types (color bars) from Velmeshev et al. (30) (rows) in the Perturb-Seq data across a panel of ASD/ND risk genes (columns). Right: DE gene expression changes in Perturb-Seq data (black dots; each dot represents an ASD/ND risk gene perturbation) compared to DE values for the 14 genes found to be DE in ASD patients in the Velmeshev et al dataset (30) (FDR&lt;0.2) (red dots). The two highlighted genes, SST and NRN1, showed decreased expression in the Perturb-Seq data (FDR&lt;0.1), consistent with the ASD patient dataset. 
         FIGS. 5A-5D —( FIG. 5A ) The frequency of de novo loss-of-function variants in ascertained Autism Spectrum Disorders (ASD) and neurodevelopmental delay (NDD) cases for the 35 risk-associated genes included the Perturb-Seq analysis. Q-value was calculated based on the de novo and case control (dncc) data. This data comes from Satterstrom et al (30). ( FIGS. 5B-5D ) Gene expression of a panel of selected ASD/ND de novo risk genes in human somatosensory cortex (S1C), striatum, and thalamus across the Allen Brain Atlas BrainSpan postmortem samples. Dendrogram indicates hierarchical clustering by rows. 
         FIGS. 6A-6C —( FIGS. 6A-6B ) Cell type clusters from E18.5 (public data from 10× Genomics) and WT P7 (data generated from this work) neocortex, as well as expression of cell-type marker genes across identified cell clusters. ( FIG. 6C ) Expression of the 38 initially-selected risk-associated genes in the cell clusters from E18.5 and P7 wild-type cortex. 
         FIGS. 7A-7G —( FIGS. 7A-7B ) Lentiviral injection at E12.5 sparsely infects neurons (NeuN+), astrocytes (Glutamine Synthase [GS]+), oligodendrocyte precursor cells (PDGFRA+), and microglia and macrophages (IBA1+) in the P7 neocortex (indicated by white arrows). Scale bar is 50 In vivo Perturb-Seq lentiviral vector with an mCherry expression cassette allows immunohistochemical identification of the targeted cell types. Lentiviral vector expression, indicated by BFP expression as well as perturbation barcode expression, was present in microglia (and likewise in all cell types included in this study,  FIGS. 8E-8F ). ( FIG. 7C ) The proportion of live cells after FACS purification is 78.2%, and &lt;0.1% of total dissociated cortical cells are BFP+ (indicated by polygons). ( FIGS. 7D-7E ) Frameshift insertion/deletion rates of the targeted loci by CRISPR/Cas9 genome editing ( FIG. 7D ) in the infected cells in vivo, and ( FIG. 7E ) in mouse embryonic stem cells in vitro as a control, for each gRNA. ( FIG. 7F ) Distribution of the perturbed cells in the 5 major cell types, across 17 different libraries (independent experimental batches) (left) and 35 different perturbation groups (right). ( FIG. 7G ) Number of genes detected in each cell type in the Perturb-Seq single-cell RNA-seq data. Quality control cutoffs for each cell type are marked by black vertical bars. 
         FIGS. 8A-8G —( FIG. 8A ) The distribution of each perturbation vector in the lentiviral pool. ( FIG. 8B ) The distribution of cell numbers from each ASD/ND perturbation group. ( FIG. 8C ) Estimated doublet score in the Perturb-Seq data using the Scrublet package; the black vertical bar represents the cutoff above which a “cell” is declared as a doublet. ( FIG. 8D ) The distribution of the number of perturbation barcodes detected per cell. ( FIG. 8E ) BFP is one of the genes with the highest expression level, detected in all 5 cell types. ( FIG. 8F ) BFP expression level is correlated with the number of genes detected in each cell type. ( FIG. 8G ) Percentage of UMI from reads mapping to the mitochondrial genome in each cell type. 
         FIGS. 9A-9C —( FIG. 9A ) Proportion of the 5 major cell types in each perturbation group. ( FIG. 9B ) Poisson regression for differences of cell type composition compared to the GFP control group. The size of the dots corresponds to base 10 log (P-value), the color to effect size. ( FIG. 9C ) Nonparametric ANOVA analysis shows that perturbation status overall (as opposed to the status of individual target genes) explains a significant portion of the variation in one glial module, ODC1. 
         FIGS. 10A-10F —( FIGS. 10A-10E ) Subclusters of each major cell class and feature plots of scores of gene modules identified by WGCNA, labelled by associated cell subtypes or biological processes. ( FIG. 10F ) Expression of key cell type marker genes in each subtype. 
         FIGS. 11A-11E —Topics identified by structural topic modeling (STM) and their correlation with WGCNA modules. Gene score indicates the lift score from STM analysis; a gene with high gene score is highly representative of the given topic. 
         FIGS. 12A-12E —Graph visualization of the 14 WGCNA network modules in 5 major cell types. Pairwise correlation was computed between each two genes, and a directed 11 nearest neighbor graph was generated and plotted with igraph. 
         FIGS. 13A-13E —( FIG. 13A ) ASD/ND risk gene perturbation effects in different WGCNA gene modules compared to GFP controls, measured by TPM. Dot color corresponds to effect size, dot size corresponds to base 10 log(P-value). P-values were extracted from the analysis in  FIGS. 3A-3F ; Padj was calculated using Benjamini &amp; Hochberg FDR correction. ( FIGS. 13B-13C ) ASD/ND risk gene perturbation effects in different WGCNA gene modules compared to GFP controls, measured as  FIG. 2B , with an alternative method for calculating P-values: instead of using the naive P-value output by the linear model (as  FIG. 2B ), they were calculated using a permutation test ( FIG. 13B ) or through a linear mixed model ( FIG. 13C ). Padj was calculated using Benjamini &amp; Hochberg FDR correction. Dot color corresponds to effect size, dot size corresponds to base 10 log(P-value). ( FIG. 13D ) Correlation of the Perturb-Seq effect size measured by TPM ( FIG. 13A ) and those measured by scaled eigen score (in  FIG. 2B ). ( FIG. 2E ) Correlation of the reported P-values generated by a linear model (in  FIG. 2B ) and those measured by a permutation test (in  FIG. 13B ). 
         FIGS. 14A-14E —( FIG. 14A ) Schematics of simplex Perturb-Seq of the ASD/ND risk gene Ank2 and a GFP control. ( FIG. 14B ) Cell type clusters from P7 neocortical simplex Ank2 Perturb-Seq. ( FIGS. 14C-14D ) Subtype clusters of inhibitory neurons from the simplex Ank2 Perturb-Seq. ( FIG. 14E ) Simplex dataset expression of the gene module IN1 identified in the pooled Perturb-Seq analysis. 
         FIGS. 15A-15D —( FIG. 15A ) ASD/ND risk gene perturbation effects in gene module ODC1 compared to GFP controls, measured by change in log TPM. ( FIGS. 15B-15C ) In situ hybridization for Pdgfra, a marker of oligodendrocyte precursor cells, in the somatosensory cortex of P7 Chd8+/− animals and wild-type littermates. Dotted lines in  FIG. 15C  indicate individual Pdgfra-positive nuclei at higher magnification (white boxes in panel  FIG. 15B ). Scale bar is 100 μm (panel  FIG. 15B ) and 10 μm (panel  FIG. 15C ), respectively. ( FIG. 15D ) Quantification of Pdgfra expression in somatosensory cortex of Chd8+/− and wild-type littermates. Each dot represents the gene expression measurement from one cell; error bars represent standard error of the mean. n=3 animals per genotype. 
         FIGS. 16A-16E —( FIG. 16A ) Cell type composition in each human brain and human brain organoid dataset. ( FIGS. 16B-16C ) Metrics used for human conservation analysis, repeated on mouse cells as a control (compare  FIGS. 4A-4B ). ( FIG. 16B ) Percent of genes in each gene module expressed in at least 5% of cells in the P7 mouse brain nuclei, Perturb-Seq dataset (whole cell), and simplex Perturb-Seq (whole cell) scRNA-seq datasets. ( FIG. 16C ) Normalized average pairwise correlation of gene expression within each gene module in each mouse dataset. Bars represent 95% confidence intervals. ( FIGS. 16D-16E ) Gene expression and modularity analyses analogous to  FIGS. 4A-4B , performed on the non-associated cell types of each module as a control, shows a lower proportion of comparisons with significant correlation coefficients and a much lower strength of correlations than in the associated cell types ( FIGS. 4A-4B ). 
         FIGS. 17A-17E —Module expression over developmental time in human brain tissues across regions (BrainSpan data) in 5 major cell types. 
         FIG. 18 —ASD/ND risk gene perturbation effects in the’ Perturb-Seq data (compared to GFP control) on 14 gene modules from the PsychEncode study comparing ASD patient and control brain tissue. Dot color corresponds to effect size, dot size corresponds to base 10 log(P-value). Padj was calculated using Benjamini &amp; Hochberg FDR correction. 
     
    
    
     The figures herein are for illustrative purposes only and are not necessarily drawn to scale. 
     DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS 
     General Definitions 
     Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F. M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E. A. Greenfield ed.); Animal Cell Culture (1987) (R. I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &amp; Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley &amp; Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011). 
     As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. 
     The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. 
     The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. 
     The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +1-10% or less, +/−5% or less, +/−1% or less, and +/−0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed. 
     As used herein, a “biological sample” may contain whole cells and/or live cells and/or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures. 
     The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. 
     Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination. 
     All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference. 
     Overview 
     Embodiments disclosed herein provide methods and uses thereof for in vivo evaluation of functions for a plurality of genes in parallel in a cell type-specific manner. The invention used a combination of in vivo genetic perturbation of progenitor cells and single-cell transcriptomic profiling of distinct types of progeny cells to interrogate the functions of multiple genes simultaneously in an in vivo environment. One of the many advantages of the present invention is that multiple genes can be evaluated simultaneously for their functions. Another major advantage of the present invention is that the function of genes in different cell types can be analyzed and revealed. Using these methods, the inventors extracted cell type-specific gene signatures for autism spectrum disorders (ASD) risk-associated genes and surprisingly found that the developmental maturation of two broad glial classes, oligodendrocyte and astrocyte are affected by loss of function mutation of selected ASD risk-associated genes. 
     In some embodiments, methods for in vivo genetic perturbation in Cas animal model are disclosed. The in vivo genetic perturbation of multiple genes in parallel described herein provides an effective way for investigating the in vivo function of genes on a large scale with single-cell resolution. The methods disclosed herein include introduction and expression of two or more guide RNAs (gRNAs) for each target gene, where each gRNA is under the control of different promoters. In some embodiments, one gRNA is under the control of mouse pol III promoter (e.g., U6) and another is under the control of human pol III promoter (e.g., U6). Additionally, the perturbations introduced into each cell are linked to a constitutively expressed reporter gene and barcode via linking the two or more gRNAs used per target gene to the reporter gene and barcode. Generally, in some embodiments, after introducing perturbations into cells (e.g., progenitor cells), cells expressing the reporter gene (which is a proxy for the presence of the gRNAs and thus the corresponding perturbations) can be enriched using a suitable technique capable of detecting reporter gene expression and separating reporter gene expressing cells from non-expressing cells to obtain an enriched perturbed cell population. Suitable sequencing and/or other analytic techniques are used to identify cell types and gene modules in the perturbed cell population that covary within a cell type and are indicative of a disease or disease state. 
     In some embodiments, the method includes delivering a pool of engineered virus particles to cells of the Cas9 animal model. In some embodiments, delivery is in utero. The pool of virus particles contains equal representation of each gRNA combination used. In this way the pool of virus particles contains equal representation of each perturbation. In some embodiments, engineered virus particle pool is generated using a suitable viral vector system (e.g., lentiviral vector system) to generate engineered virus particles (e.g., lentiviral particles) containing packaged perturbation constructs for each set of gRNAs. After packaging and generating virus particles for each set of gRNAs (and thus each perturbation construct), equal amounts or titers of each different engineered virus are combined to for the pool of engineered virus particles to be delivered. The specific perturbation construct packaged in any specific virus particle or contained in any viral vector is, in some embodiments, verified using a suitable sequencing technique prior to pooling. In some embodiments, each target gene has equal representation in the virus particle pool. In some embodiments, the suitable viral vector system includes a vector containing a two or more gRNAs, each operatively coupled to a different pol III promoter; a reporter gene and a barcode, where the reporter gene, the barcode, or both are operatively coupled to the two or more gRNAs and a constitutive pol III promoter. 
     The design, preparation, and/or utilization of the perturbation constructs herein surprisingly provides substantial superiority over the conventional methods that use single gRNA for each target gene and/or generate viral delivery particles for delivering perturbation constructs by pooling the perturbation constructs prior to viral packaging and particle generation or using an array technique. 
     In some embodiments, methods for in vivo delivering genetic perturbation are disclosed. One of the main advantages of these methods is that the genetic perturbations are delivered in parallel into progenitor cells in vivo in an animal. The progenitor cells can develop into a diversity of distinct type of progeny cells. Therefore, the present invention provides a surprising unique avenue for evaluating gene functions in each of the cell types so that the function of each gene in a plurality of genes can be interrogated in multiple cell types in parallel. 
     In some embodiments, methods for evaluating disease risk-associated genes in vivo are disclosed. One of the main advantages of the present invention is that it provides methods that the point-of-action of a plurality of risk-associated genes for a disease can be interrogated in parallel and in a cell type-specific manner. A disease commonly involves malfunction of many distinct types of cells. Therefore, the capability of the methods disclosed herein in deciphering multiple risk-associated genes of a disease in parallel in different cell types provide an innovative and effective way for functional analysis of the point-of-action of multiple risk-associated genes in vivo. 
     In some embodiments, methods for identifying therapeutic targets for a disease are disclosed. The therapeutic targets identified using the methods provided herein can more reliably represent authentic changes in molecular machinery and the cell state, thus providing attractive modality for being used for screening and evaluating drugs that are capable of treating the disease through acting on the targets. 
     Methods and Uses for In Vivo Evaluation of Gene Function and Uses Thereof 
     In some embodiments, provided are methods and uses of evaluating the functions of a plurality of genes in vivo in parallel in an animal, in which the function for each gene of interest is analyzed in multiple distinct types of cells. 
     In some embodiments, the number of plurality of genes to be evaluated in the methods disclosed herein can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10, more than 20, more than 30, more than 40, more than 50, or more than 100, more than 200, or more than 500. 
     In some embodiments, the progenitor cells that receive genetic perturbation can be 1, 2, 3, more than 3, more than 10, more than 100, more than 1000, more than 10,000, more than 100,000, more than 1 million, more than 10 million, more than 100 million, or more than 1 billion. 
     As used herein, a genetic perturbation is defined as an alteration of the structure and/or functions of a gene or gene expression products in a biological system including, but not limited to, a cell, a cell-free system, an organism, a plant, an animal, or a human. 
     Described in certain example embodiments herein are methods of identifying functions of a plurality of genes in parallel in vivo, comprising:
         a. introducing, in vivo, a plurality of genetic perturbations in each of a plurality of progenitor cells in a Cas animal model, wherein each genetic perturbation is operatively coupled to a reporter gene and a barcode;   b. generating an enriched perturbed cell population by enriching for cells expressing the reporter;   c. identifying cell types and corresponding perturbations via scRNA-seq in the enriched perturbed cell population; and   d. detecting one or more gene modules that co-vary within a cell type in the enriched perturbed cell population.       

     In certain example embodiments, identifying further comprises a genomic analysis, an epigenomic analysis, a transcriptomic analysis, a proteomic analysis, or a combination thereof. 
     In certain example embodiments, the method further comprises a genomic analysis, an epigenomic analysis, a transcriptomic analysis, a proteomic analysis, or a combination thereof. 
     In certain example embodiments, the plurality of genes are autism spectrum disorder associated genes. 
     In some embodiments, the genetic perturbation can be achieved by RNA interference, by a CRISPR-Cas system, by zinc finger nucleases (ZFN) system, by transcription-activator-like effector nucleases (TALENs) system, by short-hairpin RNA method, by gene knock-out, or by any other technologies that can introduce insertion/deletion frameshift mutations into a gene. In some embodiments, the genetic perturbation disclosed herein employs a CRISPR-Cas system. 
     Cas Animal Models 
     In some embodiments, the animal model is a Cas animal model. As used herein, the term “Cas animal model” refers to transgenic animal models that are engineered to express, either constitutively or inducibly, in one or more of their cells. This term includes progeny (including embryos) of a Cas animal model and cells thereof. In some embodiments, all of the cells of a Cas animal model contain a Cas protein gene. In some embodiments, some of the cells of a Cas animal model contain a Cas protein gene. In some embodiments, all of the cells of a Cas animal model express a Cas protein. In some embodiments, some of the cells of a Cas animal model express a Cas protein. In certain example embodiments, the Cas animal model constitutively or inducibly expresses a Cas protein in one of, a plurality of, or all of its cells. In certain example embodiments, the Cas protein is a Cas Type I, II, III, IV, or V protein. The Cas protein can be functional within a CRISPR-Cas system, of which components thereof can be provided separate from the Cas protein expressing cell, such as by viral or other delivery. In some embodiments, gRNAs are provided to the Cas animal model so as to form a complete CRISPR-Cas system. 
     CRISPR-Cas Systems and Components Thereof. 
     In general, a CRISPR-Cas or CRISPR system as used herein and in other documents, such as WO 2014/093622 (PCT/US2013/074667), refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or “RNA(s)” as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g., CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). See, e.g., Shmakov et al. (2015) “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell, DOI: dx.doi.org/10.1016/j.molcel.2015.10.008. 
     CRISPR-Cas systems can generally fall into two classes based on their architectures of their effector molecules, which are each further subdivided by type and subtype. The two classes are Class 1 and Class 2. Class 1 CRISPR-Cas systems have effector modules composed of multiple Cas proteins, some of which form crRNA-binding complexes, while Class 2 CRISPR-Cas systems include a single, multi-domain crRNA-binding protein. 
     In some embodiments, the CRISPR-Cas system that can be used to modify a polynucleotide of the present invention described herein can be a Class 1 CRISPR-Cas system. In some embodiments, the CRISPR-Cas system that can be used to modify a polynucleotide of the present invention described herein can be a Class 2 CRISPR-Cas system. 
     Class 1 CRISPR-Cas Systems 
     In some embodiments, the CRISPR-Cas system that can be used to modify a polynucleotide of the present invention described herein can be a Class 1 CRISPR-Cas system. Class 1 CRISPR-Cas systems are divided into types I, II, and IV. Makarova et al. 2020. Nat. Rev. 18: 67-83., particularly as described in  FIG. 1 . Type I CRISPR-Cas systems are divided into 9 subtypes (I-A, I-B, I-C, I-D, I-E, I-F1, I-F2, I-F3, and IG). Makarova et al., 2020. Class 1, Type I CRISPR-Cas systems can contain a Cas3 protein that can have helicase activity. Type III CRISPR-Cas systems are divided into 6 subtypes (III-A, III-B, III-E, and III-F). Type III CRISPR-Cas systems can contain a Cas10 that can include an RNA recognition motif called Palm and a cyclase domain that can cleave polynucleotides. Makarova et al., 2020. Type IV CRISPR-Cas systems are divided into 3 subtypes. (IV-A, IV-B, and IV-C). Makarova et al., 2020. Class 1 systems also include CRISPR-Cas variants, including Type I-A, I-B, I-E, I-F and I-U variants, which can include variants carried by transposons and plasmids, including versions of subtype I-F encoded by a large family of Tn7-like transposon and smaller groups of Tn7-like transposons that encode similarly degraded subtype I-B systems. Peters et al., PNAS 114 (35) (2017); DOI: 10.1073/pnas.1709035114; see also, Makarova et al. 2018. The CRISPR Journal, v. 1, n5,  FIG. 5 . 
     The Class 1 systems typically use a multi-protein effector complex, which can, in some embodiments, include ancillary proteins, such as one or more proteins in a complex referred to as a CRISPR-associated complex for antiviral defense (Cascade), one or more adaptation proteins (e.g., Cas1, Cas2, RNA nuclease), and/or one or more accessory proteins (e.g., Cas 4, DNA nuclease), CRISPR associated Rossman fold (CARF) domain containing proteins, and/or RNA transcriptase. 
     The backbone of the Class 1 CRISPR-Cas system effector complexes can be formed by RNA recognition motif domain-containing protein(s) of the repeat-associated mysterious proteins (RAMPs) family subunits (e.g., Cas 5, Cas6, and/or Cas7). RAMP proteins are characterized by having one or more RNA recognition motif domains. In some embodiments, multiple copies of RAMPs can be present. In some embodiments, the Class I CRISPR-Cas system can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more Cas5, Cas6, and/or Cas 7 proteins. In some embodiments, the Cas6 protein is an RNAse, which can be responsible for pre-crRNA processing. 
     When present in a Class 1 CRISPR-Cas system, Cash can be optionally physically associated with the effector complex. 
     Class 1 CRISPR-Cas system effector complexes can, in some embodiments, also include a large subunit. The large subunit can be composed of or include a Cas8 and/or Cas10 protein. See, e.g.,  FIGS. 1 and 2 . Koonin E V, Makarova K S. 2019. Phil. Trans. R. Soc. B 374: 20180087, DOI: 10.1098/rstb.2018.0087 and Makarova et al. 2020. 
     Class 1 CRISPR-Cas system effector complexes can, in some embodiments, include a small subunit (for example, Cash 1). See, e.g.,  FIGS. 1 and 2 . Koonin E V, Makarova K S. 2019 Origins and Evolution of CRISPR-Cas systems. Phil. Trans. R. Soc. B 374: 20180087, DOI: 10.1098/rstb.2018.0087. 
     In some embodiments, the Class 1 CRISPR-Cas system can be a Type I CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-A CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-B CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-C CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-D CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-E CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-F1 CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-F2 CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-F3 CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-G CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a CRISPR Cas variant, such as a Type I-A, I-B, I-E, I-F and I-U variants, which can include variants carried by transposons and plasmids, including versions of subtype I-F encoded by a large family of Tn7-like transposon and smaller groups of Tn7-like transposons that encode similarly degraded subtype I-B systems as previously described. 
     In some embodiments, the Class 1 CRISPR-Cas system can be a Type III CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-A CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-B CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-C CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-D CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-E CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-F CRISPR-Cas system. 
     In some embodiments, the Class 1 CRISPR-Cas system can be a Type IV CRISPR-Cas-system. In some embodiments, the Type IV CRISPR-Cas system can be a subtype IV-A CRISPR-Cas system. In some embodiments, the Type IV CRISPR-Cas system can be a subtype IV-B CRISPR-Cas system. In some embodiments, the Type IV CRISPR-Cas system can be a subtype IV-C CRISPR-Cas system. 
     The effector complex of a Class 1 CRISPR-Cas system can, in some embodiments, include a Cas3 protein that is optionally fused to a Cas2 protein, a Cas4, a Cas5, a Cash, a Cas7, a Cas8, a Cas10, a Cas11, or a combination thereof. In some embodiments, the effector complex of a Class 1 CRISPR-Cas system can have multiple copies, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, of any one or more Cas proteins. 
     Class 2 CRISPR-Cas Systems 
     The compositions, systems, and methods described in greater detail elsewhere herein can be designed and adapted for use with Class 2 CRISPR-Cas systems. Thus, in some embodiments, the CRISPR-Cas system is a Class 2 CRISPR-Cas system. Class 2 systems are distinguished from Class 1 systems in that they have a single, large, multi-domain effector protein. In certain example embodiments, the Class 2 system can be a Type II, Type V, or Type VI system, which are described in Makarova et al. “Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants” Nature Reviews Microbiology, 18:67-81 (February 2020), incorporated herein by reference. Each type of Class 2 system is further divided into subtypes. See Markova et al. 2020, particularly at Figure. 2. Class 2, Type II systems can be divided into 4 subtypes: II-A, II-B, II-C1, and II-C2. Class 2, Type V systems can be divided into 17 subtypes: V-A, V-B1, V-B2, V-C, V-D, V-E, V-F1, V-F1 (V-U3), V-F2, V-F3, V-G, V-H, V-I, V-K (V-U5), V-U1, V-U2, and V-U4. Class 2, Type IV systems can be divided into 5 subtypes: VI-A, VI-B1, VI-B2, VI-C, and VI-D. 
     The distinguishing feature of these types is that their effector complexes consist of a single, large, multi-domain protein. Type V systems differ from Type II effectors (e.g., Cas9), which contain two nuclear domains that are each responsible for the cleavage of one strand of the target DNA, with the HNH nuclease inserted inside the Ruv-C like nuclease domain sequence. The Type V systems (e.g., Cas12) only contain a RuvC-like nuclease domain that cleaves both strands. Type VI (Cas13) are unrelated to the effectors of Type II and V systems and contain two HEPN domains and target RNA. Cas13 proteins also display collateral activity that is triggered by target recognition. Some Type V systems have also been found to possess this collateral activity with two single-stranded DNA in in vitro contexts. 
     In some embodiments, the Class 2 system is a Type II system. In some embodiments, the Type II CRISPR-Cas system is a II-A CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a II-B CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a II-C1 CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a II-C2 CRISPR-Cas system. In some embodiments, the Type II system is a Cas9 system. In some embodiments, the Type II system includes a Cas9. 
     In some embodiments, the Class 2 system is a Type V system. In some embodiments, the Type V CRISPR-Cas system is a V-A CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-B1 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-B2 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-C CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-D CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-E CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-F1 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-F1 (V-U3) CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-F2 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-F3 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-G CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-H CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-I CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-K (V-U5) CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-U1 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-U2 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-U4 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system includes a Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), CasY(Cas12d), CasX (Cas12e), Cas14, and/or CasΦ. 
     Guide RNAs (gRNAs) 
     As previously described, when a CRISPR-Cas system is used to generate genetic perturbations, such as in the context of a Cas animal model, the gRNAs for the desired perturbations are subsequently delivered to cells containing a Cas protein or capable of expressing a Cas protein in the animal model. In some embodiments, a plurality of guide RNAs (gRNAs) are used for targeting a gene of interest. In certain example embodiments, the plurality of genetic perturbations are introduced using two or more guide RNAs (gRNAs) for each target gene. In some embodiments, the two or more gRNAs each bind to sequence of an exon, an intron, or both at the 5′ end of a target gene. In some embodiments, the two or more gRNAs each bind to sequences of one or more coding exons at the 5′ end of a target gene. 
     The terms guide molecule, guide sequence and guide polynucleotide, refer to polynucleotides capable of guiding Cas to a target genomic locus and are used interchangeably as in foregoing cited documents such as WO 2014/093622 (PCT/US2013/074667). In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. The guide molecule can be a polynucleotide. 
     The ability of a guide sequence (within a nucleic acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence may be assessed by any suitable assay. For example, the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay (Qui et al. 2004. BioTechniques. 36(4)702-707). Similarly, cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible and will occur to those skilled in the art. 
     A guide sequence, and hence a nucleic acid-targeting guide may be selected to target any target nucleic acid sequence. The target sequence may be DNA. The target sequence may be any RNA sequence. In some embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmatic RNA (scRNA). In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA, and lncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule. 
     In some embodiments, a nucleic acid-targeting guide is selected to reduce the degree secondary structure within the nucleic acid-targeting guide. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and P A Carr and G M Church, 2009, Nature Biotechnology 27(12): 1151-62). 
     In certain embodiments, a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence. In certain embodiments, the guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence. In certain embodiments, the direct repeat sequence may be located upstream (i.e., 5′) from the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3′) from the guide sequence or spacer sequence. 
     In certain embodiments, the crRNA comprises a stem loop, preferably a single stem loop. In certain embodiments, the direct repeat sequence forms a stem loop, preferably a single stem loop. 
     In certain embodiments, the spacer length of the guide RNA is from 15 to 35 nt. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides. In certain embodiments, the spacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27 nt, from 27 to 30 nt, e.g., 27, 28, 29, or 30 nt, from 30 to 35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer. 
     The “tracrRNA” sequence or analogous terms includes any polynucleotide sequence that has sufficient complementarity with a crRNA sequence to hybridize. In some embodiments, the degree of complementarity between the tracrRNA sequence and crRNA sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and crRNA sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. 
     In general, degree of complementarity is with reference to the optimal alignment of the sca sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the sca sequence or tracr sequence. In some embodiments, the degree of complementarity between the tracr sequence and sca sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. 
     In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence can be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length; and tracr RNA can be 30 or 50 nucleotides in length. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. Off target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementarity between the sequence and the guide, with it advantageous that off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between the sequence and the guide. 
     In some embodiments according to the invention, the guide RNA (capable of guiding Cas to a target locus) may comprise (1) a guide sequence capable of hybridizing to a genomic target locus in the eukaryotic cell; (2) a tracr sequence; and (3) a tracr mate sequence. All (1) to (3) may reside in a single RNA, i.e., an sgRNA (arranged in a 5′ to 3′ orientation), or the tracr RNA may be a different RNA than the RNA containing the guide and tracr sequence. The tracr hybridizes to the tracr mate sequence and directs the CRISPR/Cas complex to the target sequence. Where the tracr RNA is on a different RNA than the RNA containing the guide and tracr sequence, the length of each RNA may be optimized to be shortened from their respective native lengths, and each may be independently chemically modified to protect from degradation by cellular RNase or otherwise increase stability. 
     Many modifications to guide sequences are known in the art and are further contemplated within the context of this invention. Various modifications may be used to increase the specificity of binding to the target sequence and/or increase the activity of the Cas protein and/or reduce off-target effects. Example guide sequence modifications are described in PCT US2019/045582, specifically paragraphs [0178]-[0333]. which is incorporated herein by reference. 
     In some embodiments, the gRNAs are designed based on www.benchling.com. The gRNAs can also be designed using other technologies and strategies. In some embodiments, two gRNAs are used for targeting a gene of interest. In some embodiments, the two gRNAs have the same sequences. In some embodiments, the two gRNAs have different sequences. In some embodiments, the number of gRNAs for targeting a gene of interest can be 2, 3, more than 3, more than 5, or more than 10. In some embodiments, the sequences of gRNAs are the same. In some embodiments, the sequences of gRNAs are different from each other. 
     In certain example embodiments, each of the two or more gRNAs are controlled by a different pol III promoter. In some embodiments, the pol III promoters differ by organism optimization (e.g., human, mouse, chicken, dog, pig, fish, non-human primate, etc.). In some embodiments, the pol III promoters differ by type (e.g., H1, U6, 7SK, etc.). In some embodiments, the pol III promoters differ by organism optimization and type. 
     In certain example embodiments, a first gRNA of the two or more gRNAs is controlled by a human pol III promoter and a second gRNA of the two or more gRNAs is controlled by a non-human pol III promoter. In certain example embodiments, the human pol III promoter and the non-human pol III promoters are each independently selected from a U6, a 7SK, or an H1 promoter. 
     In some embodiments, the same promoter is used to control the expression of the gRNAs. In some embodiments, different promoter is used for controlling different gRNAs. In some embodiments, a mouse U6 promoter is used for controlling one gRNA expression, and a human U6 promoter is used for controlling another gRNA expression. In some embodiments, all of the gRNAs&#39; expressions are controlled by either a mouse U6 promoter or a human U6 promoter. 
     In certain example embodiments, one or more of the poll II promoters are constitutive. Thus, in any cell where the gRNAs are present, they will be expressed irrespective of temporal, spatial, and/or environmental control. When combined in a cell expressing a Cas protein, the gRNAs present can generate the genomic perturbations. It will be appreciated that when present in a cell that contains a Cas encoding sequence under control of an inducible promoter, perturbation can be controlled via control of the expression of the Cas protein in an inducible manner. Thus, in some of these embodiments, perturbation temporal and/or spatial incorporation can be controlled in vivo by controlling the on/off status of the Cas protein. This can be achieved a variety of ways and is dependent on the specific design of the inducible promoter and system. Inducible promoters are described in greater detail elsewhere herein. 
     In certain example embodiments, one or more of the pol III promoters are inducible. In some embodiments, two gRNAs are present and both are under control of inducible promoters. By inducible controlling expression of the two gRNAs present, even in cells where there is a Cas protein is present, perturbation can still be controlled such as temporally and spatially. 
     In some embodiments, the cells of the Cas animal model contains a constitutively expressed Cas protein and the gRNAs are both under the control of constitutive pol III promoters. In some embodiments, the cells of the Cas animal model contains a constitutively expressed Cas protein and the gRNAs are both under the control of inducible pol III promoters. In some embodiments, the cells of the Cas animal model contains a constitutively expressed Cas protein and one gRNAs is under the control of a constitutive pol III promoter and one or more gRNAs are under the control of an inducible promoter. 
     In some embodiments, the cells of the Cas animal model contains an inducibly expressed Cas protein and the gRNAs are both under the control of constitutive pol III promoters. In some embodiments, the cells of the Cas animal model contains an inducibly expressed Cas protein and the gRNAs are both under the control of inducible pol III promoters. In some embodiments, the cells of the Cas animal model contains an inducibly expressed Cas protein and one gRNAs is under the control of a constitutive pol III promoter and one or more gRNAs are under the control of an inducible promoter. 
     In some embodiments, the animal model (e.g., a Cas animal model) is a mouse, a rat, or a rabbit, a non-human primate, a pig, another mammal, or avian. In some embodiments, a mouse is used. In some embodiments the animal model (e.g., a Cas animal model) is a pregnant animal model. 
     The Cas animal model may comprise a cell in a model non-human organism, a model non-human mammal that expresses a Cas protein, a mouse that expresses a Cas protein, a mouse that expresses Cpf1, a cell in vivo or a cell ex vivo or a cell in vitro (see e.g., WO 2014/093622 (PCT/US13/074667); US Patent Publication Nos. 20120017290 and 20110265198 assigned to Sangamo BioSciences, Inc.; US Patent Publication No. 20130236946 assigned to Cellectis; Platt et al., “CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling” Cell (2014), 159(2): 440-455; “Oncogenic models based on delivery and use of the crispr-cas systems, vectors and compositions” WO2014204723A1 “Delivery and use of the crispr-cas systems, vectors and compositions for hepatic targeting and therapy” WO2014204726A1; “Delivery, use and therapeutic applications of the crispr-cas systems and compositions for modeling mutations in leukocytes” WO2016049251; and Chen et al., “Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis” 2015, Cell 160, 1246-1260). 
     Target Sequences, PAMs, and PFSs 
     In some embodiments, the two or more gRNAs each bind to sequence (e.g., a target sequence) in an exon, an intron, or both at the 5′ end of a target gene. In some embodiments, the two or more gRNAs each bind to sequences of one or more coding exons at the 5′ end of a target gene. 
     As used herein, “target gene” refers to a pre-selected and non-random gene or gene product whose sequence, function, expression, activity and the like are to be modified or modulated. Target genes can be objectively chosen amongst any known genes by a set of criteria. It will be appreciated that the set of criteria will be appreciated by those of ordinary skill in the art based on many factors including, but not limited to, a disease or condition being studied, a biological pathway being studied, the age of an organism being studied, the cell type, cell state, an/or tissue being studied. Target genes can be selected from personal knowledge of a person performing a method described herein, the literature, publicly accessible databases, which can be generic (e.g., NCBI&#39;s GenBank) or be focused (such as on a specific cell type, pathway, disease, and the like) (e.g., FaCD Online, DriverDBv3, BRCA Public Database, DisGeNET, MalaCards, Gene Disease Database, eDGAR, mitoMAP, Human Variome Project, Human Gene Mutation Database, and the like), and combinations thereof. Other considerations for choosing target genes for a desired disease, condition, or state, will be appreciated by those of ordinary skill in the art. Thus, in view of the description herein it is possible for one of ordinary skill in the art to choose a target gene based on their specific interests and then implement the perturbation methods described herein by objectively determining which target genes are. Thus, it will be appreciated that the methods described herein can be applied to any target gene, whether a gene has been given such a designation as of the filing date and/or priority date of this application or not. In short, a target gene can be objectively identified by one of ordinary skill in the art at a future date and perturbed using the methods described herein. The fact that a target gene is designated as such in the future does not impede the method from being operational, enabled, or fully described as to those yet-to-be designated target genes. 
     In some embodiments, target genes are defined by a gene signature or module and can be used to generate a focused gRNA library that can be used to introduce the perturbations as described elsewhere herein. In some embodiments, systematic perturbation of target genes can be performed, such as those relevant to a particular disease, cell state, or condition. Gene expression profiling can be used to define the target genes of interest as well as perform follow-up single cell and population RNA-seq analysis. 
     In some embodiments, the target genes are autism spectrum disease associated genes. In some embodiments, the target genes are autism spectrum disease risk-associated genes. 
     Target Sequences 
     In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. A target sequence may comprise RNA polynucleotides. The term “target RNA” refers to an RNA polynucleotide being or comprising the target sequence. In other words, the target polynucleotide can be a polynucleotide or a part of a polynucleotide to which a part of the guide sequence is designed to have complementarity with and to which the effector function mediated by the complex comprising the CRISPR effector protein and a guide molecule is to be directed. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. 
     The guide sequence can specifically bind a target sequence in a target polynucleotide. The target polynucleotide may be DNA. The target polynucleotide may be RNA. The target polynucleotide can have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. or more) target sequences. The target polynucleotide can be on a vector. The target polynucleotide can be genomic DNA. The target polynucleotide can be episomal. Other forms of the target polynucleotide are described elsewhere herein. 
     The target sequence may be DNA. The target sequence may be any RNA sequence. In some embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmatic RNA (scRNA). In some preferred embodiments, the target sequence (also referred to herein as a target polynucleotide) may be a sequence within an RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA, and lncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule. 
     PAM and PFS Elements 
     PAM elements are sequences that can be recognized and bound by Cas proteins. Cas proteins/effector complexes can then unwind the dsDNA at a position adjacent to the PAM element. It will be appreciated that Cas proteins and systems that include them that target RNA do not require PAM sequences (Marraffini et al. 2010. Nature. 463:568-571). Instead, many rely on PFSs, which are discussed elsewhere herein. In certain embodiments, the target sequence should be associated with a PAM (protospacer adjacent motif) or PFS (protospacer flanking sequence or site), that is, a short sequence recognized by the CRISPR complex. Depending on the nature of the CRISPR-Cas protein, the target sequence should be selected, such that its complementary sequence in the DNA duplex (also referred to herein as the non-target sequence) is upstream or downstream of the PAM. In the embodiments, the complementary sequence of the target sequence is downstream or 3′ of the PAM or upstream or 5′ of the PAM. The precise sequence and length requirements for the PAM differ depending on the Cas protein used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). Examples of the natural PAM sequences for different Cas proteins are provided herein below and the skilled person will be able to identify further PAM sequences for use with a given Cas protein. 
     The ability to recognize different PAM sequences depends on the Cas polypeptide(s) included in the system. See e.g., Gleditzsch et al. 2019. RNA Biology. 16(4):504-517. Table 1 below shows several Cas polypeptides and the PAM sequence they recognize. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Example PAM Sequences 
               
            
           
           
               
               
               
            
               
                   
                 Cas Protein 
                 PAM Sequence 
               
               
                   
               
               
                   
                 SpCas9 
                 NGG/NRG 
               
               
                   
               
               
                   
                 SaCas9 
                 NGRRT or NGRRN 
               
               
                   
               
               
                   
                 NmeCas9 
                 NNNNGATT 
               
               
                   
               
               
                   
                 CjCas9 
                 NNNNRYAC 
               
               
                   
               
               
                   
                 StCas9 
                 NNAGAAW 
               
               
                   
               
               
                   
                 Cas12a (Cpf1) 
                 TTTV 
               
               
                   
                 (including 
                   
               
               
                   
                 LbCpf1 and 
                   
               
               
                   
                 AsCpf1) 
                   
               
               
                   
               
               
                   
                 Cas12b (C2c1) 
                 TTT, TTA, and TTC 
               
               
                   
               
               
                   
                 Cas12c (C2c3) 
                 TA 
               
               
                   
               
               
                   
                 Cas12d (CasY) 
                 TA 
               
               
                   
               
               
                   
                 Cas12e (CasX) 
                 5′-TTCN-3′ 
               
               
                   
               
            
           
         
       
     
     In a preferred embodiment, the CRISPR effector protein may recognize a 3′ PAM. In certain embodiments, the CRISPR effector protein may recognize a 3′ PAM which is 5′H, wherein H is A, C or U. 
     Further, engineering of the PAM Interacting (PI) domain on the Cas protein may allow programing of PAM specificity, improve target site recognition fidelity, and increase the versatility of the CRISPR-Cas protein, for example as described for Cas9 in Kleinstiver B P et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jul. 23; 523(7561):481-5. doi: 10.1038/nature14592. As further detailed herein, the skilled person will understand that Cas13 proteins may be modified analogously. Gao et al, “Engineered Cpf1 Enzymes with Altered PAM Specificities,” bioRxiv 091611; doi: http://dx.doi.org/10.1101/091611 (Dec. 4, 2016). Doench et al. created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. The authors showed that optimization of the PAM improved activity and also provided an on-line tool for designing sgRNAs. 
     PAM sequences can be identified in a polynucleotide using an appropriate design tool, which are commercially available as well as online. Such freely available tools include, but are not limited to, CRISPRFinder and CRISPRTarget. Mojica et al. 2009. Microbiol. 155(Pt. 3):733-740; Atschul et al. 1990. J. Mol. Biol. 215:403-410; Biswass et al. 2013 RNA Biol. 10:817-827; and Grissa et al. 2007. Nucleic Acid Res. 35:W52-57. Experimental approaches to PAM identification can include, but are not limited to, plasmid depletion assays (Jiang et al. 2013. Nat. Biotechnol. 31:233-239; Esvelt et al. 2013. Nat. Methods. 10:1116-1121; Kleinstiver et al. 2015. Nature. 523:481-485), screened by a high-throughput in vivo model called PAM-SCNAR (Pattanayak et al. 2013. Nat. Biotechnol. 31:839-843 and Leenay et al. 2016. Mol. Cell. 16:253), and negative screening (Zetsche et al. 2015. Cell. 163:759-771). 
     As previously mentioned, CRISPR-Cas systems that target RNA do not typically rely on PAM sequences. Instead, such systems typically recognize protospacer flanking sites (PFSs) instead of PAMs Thus, Type VI CRISPR-Cas systems typically recognize protospacer flanking sites (PFSs) instead of PAMs. PFSs represents an analogue to PAMs for RNA targets. Type VI CRISPR-Cas systems employ a Cas13. Some Cas13 proteins analyzed to date, such as Cas13a (C2c2) identified from Leptotrichia shahii (LShCAs13a) have a specific discrimination against G at the 3′ end of the target RNA. The presence of a C at the corresponding crRNA repeat site can indicate that nucleotide pairing at this position is rejected. However, some Cas13 proteins (e.g., LwaCAs13a and PspCas13b) do not seem to have a PFS preference. See e.g., Gleditzsch et al. 2019. RNA Biology. 16(4):504-517. 
     Some Type VI proteins, such as subtype B, have 5′-recognition of D (G, T, A) and a 3′-motif requirement of NAN or NNA. One example is the Cas13b protein identified in Bergeyella zoohelcum (BzCas13b). See e.g., Gleditzsch et al. 2019. RNA Biology. 16(4):504-517. 
     Overall Type VI CRISPR-Cas systems appear to have less restrictive rules for substrate (e.g., target sequence) recognition than those that target DNA (e.g., Type V and type II). 
     Delivery 
     The present disclosure also provides delivery systems for introducing exogenous perturbation construction herein to cells in the animal model, such as Cas animal model. A delivery system may comprise one or more delivery vehicles and/or cargos. Exemplary delivery systems and methods include those described in paragraphs [00117] to [00278] of Feng Zhang et al., (WO2016106236A1), and pages 1241-1251 and Table 1 of Lino C A et al., Delivering CRISPR: a review of the challenges and approaches, DRUG DELIVERY, 2018, VOL. 25, NO. 1, 1234-1257, which are incorporated by reference herein in their entireties. In some embodiments the cargos are one or more components of the perturbation constructs described herein such as the two or more gRNAs, reporter gene, and barcode. 
     Transduction 
     The cargos, e.g., nucleic acids and/or polypeptides, can be introduced to cells by transduction by a viral or pseudoviral particle. Methods of packaging the cargos in viral particles can be accomplished using any suitable viral vector or vector systems. Such viral vector and vector systems are described in greater detail elsewhere herein. As used in this context herein “transduction” refers to the process by which foreign nucleic acids and/or proteins are introduced to a cell (prokaryote or eukaryote) by a viral or pseudo viral particle. After packaging in a viral particle or pseudo viral particle, the viral particles can be exposed to cells (e.g., in vitro, ex vivo, or in vivo) where the viral or pseudoviral particle infects the cell and delivers the cargo to the cell via transduction. Viral and pseudoviral particles can be optionally concentrated prior to exposure to target cells. In some embodiments, the virus titer of a composition containing viral and/or pseudoviral particles can be obtained and a specific titer be used to transduce cells. 
     Vectors and Vector Systems 
     Also provided herein are vectors that can contain one or more of the perturbation constructs or components thereof described herein, such as the two or more gRNAs, reporter gene and barcode. In certain embodiments, the vector can contain one or more polynucleotides encoding one or more elements of a perturbation construct described herein. The vectors can be useful in producing bacterial, fungal, yeast, plant cells, animal cells, and transgenic animals that can express one or more components of the perturbation construct described herein. Within the scope of this disclosure are vectors containing one or more of the polynucleotide sequences described herein. One or more of the polynucleotides that are part of the perturbation construct described herein can be included in a vector or vector system. The vectors and/or vector systems can be used, for example, to express one or more of the polynucleotides in a cell, such as a producer cell, to produce perturbation construct system containing virus particles described elsewhere herein. Other uses for the vectors and vector systems described herein are also within the scope of this disclosure. In general, and throughout this specification, the term “vector” refers to a tool that allows or facilitates the transfer of an entity from one environment to another. In some contexts which will be appreciated by those of ordinary skill in the art, “vector” can be a term of art to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. 
     Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. 
     Recombinant expression vectors can be composed of a nucleic acid (e.g., a polynucleotide) of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which can be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” and “operatively-linked” are used interchangeably herein and further defined elsewhere herein. In the context of a vector, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells. These and other embodiments of the vectors and vector systems are described elsewhere herein. 
     In some embodiments, the vector can be a bicistronic vector. In some embodiments, a bicistronic vector can be used for one or more elements of the perturbation construct described herein. In some embodiments, expression of elements of the perturbation construct described herein can be driven by a ubiquitous promoter, constitutive, cell-specific promoter, inducible promoter or any permissible combination thereof. In some embodiments, expression of elements of the perturbation construct described herein can be driven by a cell-specific and/or inducible promoter. Where the element of the perturbation construct system is an RNA, its expression can be driven by a Pol III promoter, such as a U6 promoter. In some embodiments, the reporter gene expression is driven by a pol II promoter, such as EF1a, beta actin, CAG, and the like. 
     In some embodiments, a vector capable of delivering a perturbation construct or component thereof to a cell can be composed of or contain a minimal promoter operably linked to a first gRNA, and/or a second gRNA, and a second minimal promoter operably linked to a first gRNA and/or a second gRNA, and a third minimal promoter operably linked to a reporter gene and, optionally, a barcode, wherein the length of the vector sequence comprising the minimal promoters and polynucleotide sequences is less than 4.4 Kb. In an embodiment, the vector can be a viral vector. In certain embodiments, the viral vector is an is an adeno-associated virus (AAV) or an adenovirus vector. 
     In one embodiment, the invention provides a vector system comprising one or more vectors. In some embodiments, the system comprises: (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences up- or downstream (whichever applicable) of the direct repeat sequence, wherein when expressed, the one or more guide sequence(s) direct(s) sequence-specific binding of the CRISPR complex to the one or more target sequence(s) in a eukaryotic cell, wherein the CRISPR complex comprises a Cas enzyme complexed with the one or more guide sequence(s) that is hybridized to the one or more target sequence(s); and (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas enzyme, preferably comprising at least one nuclear localization sequence and/or at least one NES; wherein components (a) and (b) are located on the same or different vectors of the system. Where applicable, a tracr sequence may also be provided. 
     In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a Cas CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the CRISPR complex comprises one or more nuclear localization sequences and/or one or more NES of sufficient strength to drive accumulation of said Cas CRISPR complex in a detectable amount in or out of the nucleus of a eukaryotic cell. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, each of the guide sequences is at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, or between 16-25, or between 16-20 nucleotides in length. 
     Cell-Based Vector Amplification and Expression 
     Vectors may be introduced and propagated in a prokaryote or prokaryotic cell. In some embodiments, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g., amplifying a plasmid as part of a viral vector packaging system). The vectors can be viral-based or non-viral based. In some embodiments, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. 
     Vectors can be designed for expression of one or more elements of the perturbation construct described herein (e.g., nucleic acids, transcripts, proteins, enzymes, and combinations thereof) in a suitable host cell. In some embodiments, the suitable host cell is a prokaryotic cell. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. In some embodiments, the suitable host cell is a eukaryotic cell. 
     In some embodiments, the suitable host cell is a suitable bacterial cell. Suitable bacterial cells include, but are not limited to, bacterial cells from the bacteria of the species  Escherichia coli . Many suitable strains of  E. coli  are known in the art for expression of vectors. These include, but are not limited to Pir1, Stb12, Stb13, Stb14, TOP10, XL1 Blue, and XL10 Gold. In some embodiments, the host cell is a suitable insect cell. Suitable insect cells include those from  Spodoptera frugiperda . Suitable strains of  S. frugiperda  cells include, but are not limited to, Sf9 and Sf21. In some embodiments, the host cell is a suitable yeast cell. In some embodiments, the yeast cell can be from  Saccharomyces cerevisiae . In some embodiments, the host cell is a suitable mammalian cell. Many types of mammalian cells have been developed to express vectors. Suitable mammalian cells include, but are not limited to, HEK293, Chinese Hamster Ovary Cells (CHOs), mouse myeloma cells, HeLa, U205, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-Rb50, HepG G2, DIKX-X11, J558L, Baby hamster kidney cells (BHK), and chicken embryo fibroblasts (CEFs). Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). 
     In some embodiments, the vector can be a yeast expression vector. Examples of vectors for expression in yeast  Saccharomyces cerevisiae  include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). As used herein, a “yeast expression vector” refers to a nucleic acid that contains one or more sequences encoding an RNA and/or polypeptide and may further contain any desired elements that control the expression of the nucleic acid(s), as well as any elements that enable the replication and maintenance of the expression vector inside the yeast cell. Many suitable yeast expression vectors and features thereof are known in the art; for example, various vectors and techniques are illustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (Humana Press, New York, 2007) and Buckholz, R. G. and Gleeson, M. A. (1991) Biotechnology (NY) 9(11): 1067-72. Yeast vectors can contain, without limitation, a centromeric (CEN) sequence, an autonomous replication sequence (ARS), a promoter, such as an RNA Polymerase III promoter, operably linked to a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., auxotrophic, antibiotic, or other selectable markers). Examples of expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and episomal plasmids. 
     In some embodiments, the vector is a baculovirus vector or expression vector and can be suitable for expression of polynucleotides and/or proteins in insect cells. In some embodiments, the suitable host cell is an insect cell. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39). rAAV (recombinant Adeno-associated viral) vectors are preferably produced in insect cells, e.g.,  Spodoptera frugiperda  Sf9 insect cells, grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial vendors, e.g., Sigma Aldrich (EX-CELL 405). 
     In some embodiments, the vector is a mammalian expression vector. In some embodiments, the mammalian expression vector is capable of expressing one or more polynucleotides and/or polypeptides in a mammalian cell. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). The mammalian expression vector can include one or more suitable regulatory elements capable of controlling expression of the one or more polynucleotides and/or proteins in the mammalian cell. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. More detail on suitable regulatory elements are described elsewhere herein. 
     For other suitable expression vectors and vector systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. 
     In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987 . Genes Dev.  1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988 . Adv. Immunol.  43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989 . EMBO J.  8: 729-733) and immunoglobulins (Baneiji, et al., 1983 . Cell  33: 729-740; Queen and Baltimore, 1983 . Cell  33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989 . Proc. Natl. Acad. Sci. USA  86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985 . Science  230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990 . Science  249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989 . Genes Dev.  3: 537-546). With regards to these prokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No. 6,750,059, the contents of which are incorporated by reference herein in their entirety. Other embodiments can utilize viral vectors, with regards to which mention is made of U.S. patent application Ser. No. 13/092,085, the contents of which are incorporated by reference herein in their entirety. Tissue-specific regulatory elements are known in the art and in this regard, mention is made of U.S. Pat. No. 7,776,321, the contents of which are incorporated by reference herein in their entirety. In some embodiments, a regulatory element can be operably linked to one or more elements of a perturbation construct described herein so as to drive expression of the one or more elements of the perturbation construct described herein. 
     In some embodiments, the vector can be a fusion vector or fusion expression vector. In some embodiments, fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus, carboxy terminus, or both of a recombinant protein. Such fusion vectors can serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. In some embodiments, expression of polynucleotides (such as non-coding polynucleotides) and proteins in prokaryotes can be carried out in  Escherichia coli  with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polynucleotides and/or proteins. In some embodiments, the fusion expression vector can include a proteolytic cleavage site, which can be introduced at the junction of the fusion vector backbone or other fusion moiety and the recombinant polynucleotide or protein to enable separation of the recombinant polynucleotide or protein from the fusion vector backbone or other fusion moiety subsequent to purification of the fusion polynucleotide or protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion  E. coli  expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). 
     In some embodiments, one or more vectors driving expression of one or more elements of a perturbation construct described herein are introduced into a host cell, such as in an animal model (e.g., a Cas animal model) such that expression of the elements of the engineered delivery system described herein direct formation a CRISPR-Cas complex at one or more target sites. For example, a CRISPR-Cas effector protein described herein can be provided in the host cell and a nucleic acid component (e.g., a guide polynucleotide) can be operably linked to a regulatory elements on separate vectors. Different or all elements of perturbation construct described herein can be delivered to an animal, plant, microorganism or cell thereof to produce an animal (e.g., a mammal, reptile, avian, etc.), plant, microorganism or cell thereof that constitutively, inducibly, or conditionally expresses all or different elements of the perturbation construct described herein. As previously described the host cell can express or be capable of expressing a Cas protein, such that when gRNAs present in the perturbation construct are expressed in the same host cell, a CRISPR-Cas system is generated, and genetic perturbations can be introduced in that cell. 
     In some embodiments, two or more of the elements expressed from the same or different regulatory element(s), can be combined in a single vector, with one or more additional vectors providing any components of the system not included in the first vector. perturbation construct polynucleotides that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5′ with respect to (“upstream” of) or 3′ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element and oriented in the same or opposite direction. 
     Vector Features 
     The vectors can include additional features that can confer one or more functionalities to the vector, the polynucleotide to be delivered, a virus particle produced there from, or polypeptide expressed thereof. Such features include, but are not limited to, regulatory elements, selectable markers, molecular identifiers (e.g., molecular barcodes), stabilizing elements, and the like. It will be appreciated by those skilled in the art that the design of the expression vector and additional features included can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. 
     Regulatory Elements 
     In certain embodiments, the polynucleotides and/or vectors thereof described herein (such as the perturbation construct of the present invention) can include one or more regulatory elements that can be operatively linked to the polynucleotide. The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences) and cellular localization signals (e.g., nuclear localization signals). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter can direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6, 7SK, and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). Specific configurations of the gRNAs, reporter gene and pol II and pol III promoters in the context of the present invention are described in greater detail elsewhere herein. 
     In some embodiments, the regulatory sequence can be a regulatory sequence described in U.S. Pat. No. 7,776,321, U.S. Pat. Pub. No. 2011/0027239, and International Patent Publication No. WO 2011/028929, the contents of which are incorporated by reference herein in their entirety. In some embodiments, the vector can contain a minimal promoter. In some embodiments, the minimal promoter is the Mecp2 promoter, tRNA promoter, or U6. In a further embodiment, the minimal promoter is tissue specific. In some embodiments, the length of the vector polynucleotide the minimal promoters and polynucleotide sequences is less than 4.4 Kb. 
     To express a polynucleotide, the vector can include one or more transcriptional and/or translational initiation regulatory sequences, e.g., promoters, that direct the transcription of the gene and/or translation of the encoded protein in a cell. In some embodiments a constitutive promoter may be employed. Suitable constitutive promoters for mammalian cells are generally known in the art and include, but are not limited to SV40, CAG, CMV, EF-1α, β-actin, RSV, and PGK. Suitable constitutive promoters for bacterial cells, yeast cells, and fungal cells are generally known in the art, such as a T-7 promoter for bacterial expression and an alcohol dehydrogenase promoter for expression in yeast. 
     In some embodiments, the regulatory element can be a regulated promoter. “Regulated promoter” refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and includes tissue-specific, tissue-preferred and inducible promoters. Regulated promoters include conditional promoters and inducible promoters. In some embodiments, conditional promoters can be employed to direct expression of a polynucleotide in a specific cell type, under certain environmental conditions, and/or during a specific state of development. Suitable tissue specific promoters can include, but are not limited to, liver specific promoters (e.g. APOA2, SERPIN A1 (hAAT), CYP3A4, and MIR122), pancreatic cell promoters (e.g. INS, IRS2, Pdx1, Alx3, Ppy), cardiac specific promoters (e.g. Myh6 (alpha MHC), MYL2 (MLC-2v), TNI3 (cTn1), NPPA (ANF), Slc8a1 (Ncx1)), central nervous system cell promoters (SYN1, GFAP, INA, NES, MOBP, MBP, TH, FOXA2 (HNF3 beta)), skin cell specific promoters (e.g. FLG, K14, TGM3), immune cell specific promoters, (e.g. ITGAM, CD43 promoter, CD14 promoter, CD45 promoter, CD68 promoter), urogenital cell specific promoters (e.g. Pbsn, Upk2, Sbp, Fer114), endothelial cell specific promoters (e.g. ENG), pluripotent and embryonic germ layer cell specific promoters (e.g. Oct4, NANOG, Synthetic Oct4, T brachyury, NES, SOX17, FOXA2, MIR122), and muscle cell specific promoter (e.g. Desmin). Other tissue and/or cell specific promoters are generally known in the art and are within the scope of this disclosure. 
     Inducible/conditional promoters can be positively inducible/conditional promoters (e.g. a promoter that activates transcription of the polynucleotide upon appropriate interaction with an activated activator, or an inducer (compound, environmental condition, or other stimulus) or a negative/conditional inducible promoter (e.g. a promoter that is repressed (e.g. bound by a repressor) until the repressor condition of the promotor is removed (e.g. inducer binds a repressor bound to the promoter stimulating release of the promoter by the repressor or removal of a chemical repressor from the promoter environment). The inducer can be a compound, environmental condition, or other stimulus. Thus, inducible/conditional promoters can be responsive to any suitable stimuli such as chemical, biological, or other molecular agents, temperature, light, and/or pH. Suitable inducible/conditional promoters include, but are not limited to, Tet-On, Tet-Off, Lac promoter, pBad, AlcA, LexA, Hsp70 promoter, Hsp90 promoter, pDawn, XVE/OlexA, GVG, and pOp/LhGR. 
     Examples of promoters that are inducible and that can allow for spatiotemporal control of gene editing or gene expression may use a form of energy. The form of energy may include but is not limited to sound energy, electromagnetic radiation, chemical energy and/or thermal energy. Examples of inducible systems include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc.), or light inducible systems (Phytochrome, LOV domains, or cryptochrome)., such as a Light Inducible Transcriptional Effector (LITE) that direct changes in transcriptional activity in a sequence-specific manner. The components of a light inducible system may include one or more elements of the CRISPR-Cas system described herein, a light-responsive cytochrome heterodimer (e.g., from  Arabidopsis thaliana ), and a transcriptional activation/repression domain. In some embodiments, the vector can include one or more of the inducible DNA binding proteins provided in International Patent Publication No. WO 2014/018423 and US Patent Publication Nos., 2015/0291966, 2017/0166903, 2019/0203212, which describe e.g., embodiments of inducible DNA binding proteins and methods of use and can be adapted for use with the present invention. 
     In some embodiments, transient or inducible expression can be achieved by including, for example, chemical-regulated promotors, i.e., whereby the application of an exogenous chemical induces gene expression. Modulation of gene expression can also be obtained by including a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters include, but are not limited to, the maize 1n2-2 promoter, activated by benzene sulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-11-27, WO93/01294), activated by hydrophobic electrophilic compounds used as pre-emergent herbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7) activated by salicylic acid. Promoters which are regulated by antibiotics, such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156) can also be used herein. 
     In some embodiments, the polynucleotide, vector or system thereof can include one or more elements capable of translocating and/or expressing a one or more elements of a perturbation construct described herein to/in a specific cell component or organelle. Such organelles can include, but are not limited to, nucleus, ribosome, endoplasmic reticulum, Golgi apparatus, chloroplast, mitochondria, vacuole, lysosome, cytoskeleton, plasma membrane, cell wall, peroxisome, centrioles, etc. Such regulatory elements can include, but are not limited to, nuclear localization signals (examples of which are described in greater detail elsewhere herein), any such as those that are annotated in the LocSigDB database (see e.g., http://genome.unmc.edu/LocSigDB/ and Negi et al., 2015. Database. 2015: bav003; doi: 10.1093/database/bav003), nuclear export signals, endoplasmic reticulum localization/retention signals (see e.g., Liu et al. 2007 Mol. Biol. Cell. 18(3):1073-1082 and Gorleku et al., 2011. J. Biol. Chem. 286:39573-39584), mitochondria (see e.g., Cell Reports. 22:2818-2826, particularly at  FIG. 2 ; Doyle et al. 2013. PLoS ONE 8, e67938; Funes et al. 2002. J. Biol. Chem. 277:6051-6058; Matouschek et al. 1997. PNAS USA 85:2091-2095; Oca-Cossio et al., 2003. 165:707-720; Waltner et al., 1996. J. Biol. Chem. 271:21226-21230; Wilcox et al., 2005. PNAS USA 102:15435-15440; Galanis et al., 1991. FEBS Lett 282:425-430, peroxisome (e.g. (S/A/C)-(K/R/H)-(L/A), SLK, (R/K)-(L/V/I)-XXXXX-(H/Q)-(L/A/F). Suitable protein targeting motifs can also be designed or identified using any suitable database or prediction tool, including but not limited to Minimotif Miner (http:minimotifminer.org, http://mitominer.mrc-mbu.cam.ac.uk/release-4.0/embodiment.do?name=Protein %20MT S), LocDB (see above), PTSs predictor ( ), TargetP-2.0 (http://www.cbs.dtu.dk/services/TargetP/), ChloroP (http://www.cbs.dtu.dk/services/ChloroP/); NetNES (http://www.cbs.dtu.dk/services/NetNES/), Predotar (https://urgi.versailles.inra.fr/predotar/), and SignalP (http://www.cbs.dtu.dk/services/SignalP/). 
     Reporter Genes, Selectable Markers, and Tags 
     In some embodiments, one or more of the gRNAs and/or barcodes of the perturbation construct described herein is operably linked, fused to, or otherwise modified to include a polynucleotide that encodes or is a selectable marker or tag, which can be a polynucleotide or polypeptide. Such configurations are described in greater detail elsewhere herein. 
     It will be appreciated that the polynucleotide encoding such selectable markers or tags can be incorporated into a polynucleotide encoding one or more components of the CRISPR-Cas system described herein in an appropriate manner to allow expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein and will be instantly appreciated by one of ordinary skill in the art in view of this disclosure. Many such selectable markers and tags are generally known in the art and are intended to be within the scope of this disclosure. 
     Reporter genes/proteins, selectable markers and tags include, but are not limited to, affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag; solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP, and GST; chromatography tags such as those consisting of polyanionic amino acids, such as FLAG-tag; epitope tags such as V5-tag, Myc-tag, HA-tag and NE-tag; protein tags that can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging), DNA and/or RNA segments that contain restriction enzyme or other enzyme cleavage sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT)) and the like; DNA and/or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and/or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as β-galactosidase, GUS; optically active proteins (e.g. fluorescent proteins such as a green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), blue (BFP) luciferase, and cell surface proteins); polynucleotides that can generate one or more new primer sites for PCR (e.g., the juxtaposition of two DNA sequences not previously juxtaposed), DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g. GFP, FLAG- and His-tags), and, DNA sequences that make a molecular barcode or unique molecular identifier (UMI), DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art. 
     In some embodiments, the reporter gene can be a gene coding for a cluster of differentiation (CD) molecule or CD molecules. The CD molecules that can be used as a reporter herein include, but are not limited to, CD3, CD4, CD8, CD19, CD20, CD22, CD27, CD29, CD30, CD33, CD34, CD44, CD45, CD47, CD48, CD58, CD66, CD70, CD79, CD80, CD82, CD86, CD101, and CD156. In some embodiments, the reporter gene can be a gene coding for a cell surface receptor that include, but are not limited to, EGFR, FGFR, HER2, and HER3. In certain example embodiments, the reporter gene encodes a cell surface molecules selected from the group of: CD3, CD4, CD19, CD20, CD22, CD34, CD45, CD80, a cell surface receptor, a cluster differentiation (CD) molecule, or any combination thereof. 
     Reporter genes, selectable markers, and tags can be operably linked to one or more components of the perturbation construct described herein via suitable linker, such as a glycine or glycine serine linkers, which are generally known in the art. Other suitable linkers are described elsewhere herein and generally known in the art. 
     The vector or vector system can include one or more polynucleotides encoding one or more targeting moieties. In some embodiments, the targeting moiety encoding polynucleotides can be included in the vector or vector system, such as a viral vector system, such that they are expressed within and/or on the virus particle(s) produced such that the virus particles can be targeted to specific cells, tissues, organs, etc. In some embodiments, the targeting moiety encoding polynucleotides can be included in the vector or vector system such that the perturbation construct described herein and/or products expressed therefrom include the targeting moiety and can be targeted to specific cells, tissues, organs, etc. In some embodiments, such as non-viral carriers, the targeting moiety can be attached to the carrier (e.g., polymer, lipid, inorganic molecule etc.) and can be capable of targeting the carrier and any attached or associated perturbation construct or component thereof described herein to specific cells, tissues, organs, etc. 
     Codon Optimization of Vector Polynucleotides 
     As described elsewhere herein, the polynucleotide encoding one or more embodiments of the perturbation construct described herein can be codon optimized. In some embodiments, one or more polynucleotides contained in a vector (“vector polynucleotides”) described herein that are in addition to an optionally codon optimized polynucleotide encoding embodiments of the perturbation construct described herein can be codon optimized. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, P A), are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a DNA/RNA-targeting Cas protein corresponds to the most frequently used codon for a particular amino acid. As to codon usage in yeast, reference is made to the online Yeast Genome database available at http://www.yeastgenome.org/community/codon_usage.shtml, or Codon selection in yeast, Bennetzen and Hall, J Biol Chem. 1982 Mar. 25; 257(6):3026-31. As to codon usage in plants including algae, reference is made to  Codon usage in higher plants, green algae, and cyanobacteria , Campbell and Gowri, Plant Physiol. 1990 January; 92(1): 1-11.; as well as  Codon usage in plant genes , Murray et al, Nucleic Acids Res. 1989 Jan. 25; 17(2):477-98; or  Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages , Morton B R, J Mol Evol. 1998 April; 46(4):449-59. 
     The vector polynucleotide can be codon optimized for expression in a specific cell-type, tissue type, organ type, and/or subject type. In some embodiments, a codon optimized sequence is a sequence optimized for expression in a eukaryote, e.g., humans (i.e., being optimized for expression in a human or human cell), or for another eukaryote, such as another animal (e.g., a mammal or avian) as is described elsewhere herein. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In some embodiments, the polynucleotide is codon optimized for a specific cell type. Such cell types can include, but are not limited to, epithelial cells (including skin cells, cells lining the gastrointestinal tract, cells lining other hollow organs), nerve cells (nerves, brain cells, spinal column cells, nerve support cells (e.g. astrocytes, glial cells, Schwann cells etc.), muscle cells (e.g. cardiac muscle, smooth muscle cells, and skeletal muscle cells), connective tissue cells (fat and other soft tissue padding cells, bone cells, tendon cells, cartilage cells), blood cells, stem cells and other progenitor cells, immune system cells, germ cells, and combinations thereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In some embodiments, the polynucleotide is codon optimized for a specific tissue type. Such tissue types can include, but are not limited to, muscle tissue, connective tissue, connective tissue, nervous tissue, and epithelial tissue. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In some embodiments, the polynucleotide is codon optimized for a specific organ. Such organs include, but are not limited to, muscles, skin, intestines, liver, spleen, brain, lungs, stomach, heart, kidneys, gallbladder, pancreas, bladder, thyroid, bone, blood vessels, blood, and combinations thereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. 
     In some embodiments, a vector polynucleotide is codon optimized for expression in particular cells, such as prokaryotic or eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as discussed herein, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. 
     Vector Construction 
     The vectors described herein can be constructed using any suitable process or technique. In some embodiments, one or more suitable recombination and/or cloning methods or techniques can be used to the vector(s) described herein. Suitable recombination and/or cloning techniques and/or methods can include, but not limited to, those described in U.S. Patent Publication No. US 2004/0171156 A1. Other suitable methods and techniques are described elsewhere herein. 
     Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &amp; Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). Any of the techniques and/or methods can be used and/or adapted for constructing an AAV or other vector described herein. nAAV vectors are discussed elsewhere herein. 
     In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and/or downstream of one or more sequence elements of one or more vectors. When multiple different guide polynucleotides are used, a single expression construct may be used to target nucleic acid-targeting activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide s polynucleotides. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-polynucleotide-containing vectors may be provided, and optionally delivered to a cell. 
     Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a CRISPR-Cas system described herein are as used in the foregoing documents, such as International Patent Publication No. WO 2014/093622 (PCT/US2013/074667) and are discussed in greater detail herein. 
     Viral Vectors 
     In some embodiments, the vector is a viral vector. The term of art “viral vector” and as used herein in this context refers to polynucleotide based vectors that contain one or more elements from or based upon one or more elements of a virus that can be capable of expressing and packaging a polynucleotide, such as a perturbation construct of the present invention, into a virus particle and producing said virus particle when used alone or with one or more other viral vectors (such as in a viral vector system). Viral vectors and systems thereof can be used for producing viral particles for delivery of and/or expression of one or more components of the perturbation construct described herein. The viral vector can be part of a viral vector system involving multiple vectors. In some embodiments, systems incorporating multiple viral vectors can increase the safety of these systems. Suitable viral vectors can include retroviral-based vectors, lentiviral-based vectors, adenoviral-based vectors, adeno associated vectors, helper-dependent adenoviral (HdAd) vectors, hybrid adenoviral vectors, herpes simplex virus-based vectors, poxvirus-based vectors, and Epstein-Barr virus-based vectors. Other embodiments of viral vectors and viral particles produce therefrom are described elsewhere herein. In some embodiments, the viral vectors are configured to produce replication incompetent viral particles for improved safety of these systems. 
     In certain embodiments, the virus structural component, which can be encoded by one or more polynucleotides in a viral vector or vector system, comprises one or more capsid proteins including an entire capsid. In certain embodiments, such as wherein a viral capsid comprises multiple copies of different proteins, the delivery system can provide one or more of the same protein or a mixture of such proteins. For example, AAV comprises 3 capsid proteins, VP1, VP2, and VP3, thus delivery systems of the invention can comprise one or more of VP1, and/or one or more of VP2, and/or one or more of VP3. Accordingly, the present invention is applicable to a virus within the family Adenoviridae, such as Atadenovirus, e.g., Ovine atadenovirus D, Aviadenovirus, e.g., Fowl aviadenovirus A, Ichtadenovirus, e.g., Sturgeon ichtadenovirus A, Mastadenovirus (which includes adenoviruses such as all human adenoviruses), e.g., Human mastadenovirus C, and Siadenovirus, e.g., Frog siadenovirus A. Thus, a virus of within the family Adenoviridae is contemplated as within the invention with discussion herein as to adenovirus applicable to other family members. Target-specific AAV capsid variants can be used or selected. Non-limiting examples include capsid variants selected to bind to chronic myelogenous leukemia cells, human CD34 PBPC cells, breast cancer cells, cells of lung, heart, dermal fibroblasts, melanoma cells, stem cell, glioblastoma cells, coronary artery endothelial cells and keratinocytes. See, e.g., Buning et al, 2015, Current Opinion in Pharmacology 24, 94-104. From teachings herein and knowledge in the art as to modifications of adenovirus (see, e.g., U.S. Pat. Nos. 9,410,129, 7,344,872, 7,256,036, 6,911,199, 6,740,525; Matthews, “Capsid-Incorporation of Antigens into Adenovirus Capsid Proteins for a Vaccine Approach,” Mol Pharm, 8(1): 3-11 (2011)), as well as regarding modifications of AAV, the skilled person can readily obtain a modified adenovirus that has a large payload protein or a CRISPR-protein, despite that heretofore it was not expected that such a large protein could be provided on an adenovirus. And as to the viruses related to adenovirus mentioned herein, as well as to the viruses related to AAV mentioned elsewhere herein, the teachings herein as to modifying adenovirus and AAV, respectively, can be applied to those viruses without undue experimentation from this disclosure and the knowledge in the art. 
     In some embodiments, the viral vector is configured such that when the cargo is packaged the cargo(s) (e.g., one or more components of the perturbation construct including but not limited to the two or more gRNAs, is/are external to the capsid or virus particle. In the sense that it is not inside the capsid (enveloped or encompassed with the capsid) but is externally exposed so that it can contact the target genomic DNA. In some embodiments, the viral vector is configured such that all the cargo(s) are contained within the capsid after packaging. 
     Retroviral and Lentiviral Vectors 
     Retroviral vectors can be composed of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Suitable retroviral vectors for the perturbation construct described herein can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). Selection of a retroviral gene transfer system may therefore depend on the target tissue. 
     The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and are described in greater detail elsewhere herein. A retrovirus can also be engineered to allow for conditional expression of the inserted transgene, such that only certain cell types are infected by the lentivirus. 
     Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells. Advantages of using a lentiviral approach can include the ability to transduce or infect non-dividing cells and their ability to typically produce high viral titers, which can increase efficiency or efficacy of production and delivery. Suitable lentiviral vectors include, but are not limited to, human immunodeficiency virus (HIV)-based lentiviral vectors, feline immunodeficiency virus (FIV)-based lentiviral vectors, simian immunodeficiency virus (SIV)-based lentiviral vectors, Moloney Murine Leukaemia Virus (Mo-MLV), Visna.maedi virus (VMV)-based lentiviral vector, carpine arthritis-encephalitis virus (CAEV)-based lentiviral vector, bovine immune deficiency virus (BIV)-based lentiviral vector, and Equine infectious anemia (EIAV)-based lentiviral vector. In some embodiments, an HIV-based lentiviral vector system can be used. In some embodiments, a FIV-based lentiviral vector system can be used. 
     In some embodiments, the lentiviral vector is an EIAV-based lentiviral vector or vector system. EIAV vectors have been used to mediate expression, packaging, and/or delivery in other contexts, such as for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8: 275-285). In another embodiment, RetinoStat®, (see, e.g., Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)), which describes RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. Any of these vectors described in these publications can be modified for the elements of the perturbation construct described herein. 
     In some embodiments, the lentiviral vector or vector system thereof can be a first-generation lentiviral vector or vector system thereof. First-generation lentiviral vectors can contain a large portion of the lentivirus genome, including the gag and pol genes, other additional viral proteins (e.g., VSV-G) and other accessory genes (e.g., vif, vprm vpu, nef, and combinations thereof), regulatory genes (e.g., tat and/or rev) as well as the gene of interest between the LTRs. First generation lentiviral vectors can result in the production of virus particles that can be capable of replication in vivo, which may not be appropriate for some instances or applications. 
     In some embodiments, the lentiviral vector or vector system thereof can be a second-generation lentiviral vector or vector system thereof. Second-generation lentiviral vectors do not contain one or more accessory virulence factors and do not contain all components necessary for virus particle production on the same lentiviral vector. This can result in the production of a replication-incompetent virus particle and thus increase the safety of these systems over first-generation lentiviral vectors. In some embodiments, the second-generation vector lacks one or more accessory virulence factors (e.g., vif, vprm, vpu, nef, and combinations thereof). Unlike the first-generation lentiviral vectors, no single second generation lentiviral vector includes all features necessary to express and package a polynucleotide into a virus particle. In some embodiments, the envelope and packaging components are split between two different vectors with the gag, pol, rev, and tat genes being contained on one vector and the envelope protein (e.g., VSV-G) are contained on a second vector. The gene of interest, its promoter, and LTRs can be included on a third vector that can be used in conjunction with the other two vectors (packaging and envelope vectors) to generate a replication-incompetent virus particle. 
     In some embodiments, the lentiviral vector or vector system thereof can be a third-generation lentiviral vector or vector system thereof. Third-generation lentiviral vectors and vector systems thereof have increased safety over first- and second-generation lentiviral vectors and systems thereof because, for example, the various components of the viral genome are split between two or more different vectors but used together in vitro to make virus particles, they can lack the tat gene (when a constitutively active promoter is included up-stream of the LTRs), and they can include one or more deletions in the 3′LTR to create self-inactivating (SIN) vectors having disrupted promoter/enhancer activity of the LTR. In some embodiments, a third-generation lentiviral vector system can include (i) a vector plasmid that contains the polynucleotide of interest and upstream promoter that are flanked by the 5′ and 3′ LTRs, which can optionally include one or more deletions present in one or both of the LTRs to render the vector self-inactivating; (ii) a “packaging vector(s)” that can contain one or more genes involved in packaging a polynucleotide into a virus particle that is produced by the system (e.g. gag, pol, and rev) and upstream regulatory sequences (e.g. promoter(s)) to drive expression of the features present on the packaging vector, and (iii) an “envelope vector” that contains one or more envelope protein genes and upstream promoters. In certain embodiments, the third-generation lentiviral vector system can include at least two packaging vectors, with the gag-pol being present on a different vector than the rev gene. 
     In some embodiments, self-inactivating lentiviral vectors with an siRNA targeting a common exon shared by HIV tat/rev, a nucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) can be used/and or adapted to the perturbation construct of the present invention. 
     In some embodiments, the pseudotype and infectivity or tropism of a lentivirus particle can be tuned by altering the type of envelope protein(s) included in the lentiviral vector or system thereof. As used herein, an “envelope protein” or “outer protein” means a protein exposed at the surface of a viral particle that is not a capsid protein. For example, envelope or outer proteins typically comprise proteins embedded in the envelope of the virus. In some embodiments, a lentiviral vector or vector system thereof can include a VSV-G envelope protein. VSV-G mediates viral attachment to an LDL receptor (LDLR) or an LDLR family member present on a host cell, which triggers endocytosis of the viral particle by the host cell. Because LDLR is expressed by a wide variety of cells, viral particles expressing the VSV-G envelope protein can infect or transduce a wide variety of cell types. Other suitable envelope proteins can be incorporated based on the host cell that a user desires to be infected by a virus particle produced from a lentiviral vector or system thereof described herein and can include, but are not limited to, feline endogenous virus envelope protein (RD114) (see e.g., Hanawa et al. Molec. Ther. 2002 5(3) 242-251), modified Sindbis virus envelope proteins (see e.g., Morizono et al. 2010. J. Virol. 84(14) 6923-6934; Morizono et al. 2001. J. Virol. 75:8016-8020; Morizono et al. 2009. J. Gene Med. 11:549-558; Morizono et al. 2006 Virology 355:71-81; Morizono et al J. Gene Med. 11:655-663, Morizono et al. 2005 Nat. Med. 11:346-352), baboon retroviral envelope protein (see e.g., Girard-Gagnepain et al. 2014. Blood. 124: 1221-1231); Tupaia paramyxovirus glycoproteins (see e.g., Enkirch T. et al., 2013. Gene Ther. 20:16-23); measles virus glycoproteins (see e.g., Funke et al. 2008. Molec. Ther. 16(8): 1427-1436), rabies virus envelope proteins, MLV envelope proteins, Ebola envelope proteins, baculovirus envelope proteins, filovirus envelope proteins, hepatitis E1 and E2 envelope proteins, gp41 and gp120 of HIV, hemagglutinin, neuraminidase, M2 proteins of influenza virus, and combinations thereof. 
     In some embodiments, the tropism of the resulting lentiviral particle can be tuned by incorporating cell targeting peptides into a lentiviral vector such that the cell targeting peptides are expressed on the surface of the resulting lentiviral particle. In some embodiments, a lentiviral vector can contain an envelope protein that is fused to a cell targeting protein (see e.g., Buchholz et al. 2015. Trends Biotechnol. 33:777-790; Bender et al. 2016. PLoS Pathog. 12(e1005461); and Friedrich et al. 2013. Mol. Ther. 2013. 21: 849-859. 
     In some embodiments, a split-intein-mediated approach to target lentiviral particles to a specific cell type can be used (see e.g., Chamoun-Emaneulli et al. 2015. Biotechnol. Bioeng. 112:2611-2617, Ramirez et al. 2013. Protein. Eng. Des. Sel. 26:215-233. In these embodiments, a lentiviral vector can contain one half of a splicing-deficient variant of the naturally split intein from  Nostoc punctiforme  fused to a cell targeting peptide and the same or different lentiviral vector can contain the other half of the split intein fused to an envelope protein, such as a binding-deficient, fusion-competent virus envelope protein. This can result in production of a virus particle from the lentiviral vector or vector system that includes a split intein that can function as a molecular Velcro linker to link the cell-binding protein to the pseudotyped lentivirus particle. This approach can be advantageous for use where surface-incompatibilities can restrict the use of, e.g., cell targeting peptides. 
     In some embodiments, a covalent-bond-forming protein-peptide pair can be incorporated into one or more of the lentiviral vectors described herein to conjugate a cell targeting peptide to the virus particle (see e.g., Kasaraneni et al. 2018. Sci. Reports (8) No. 10990). In some embodiments, a lentiviral vector can include an N-terminal PDZ domain of InaD protein (PDZ1) and its pentapeptide ligand (TEFCA) from NorpA, which can conjugate the cell targeting peptide to the virus particle via a covalent bond (e.g., a disulfide bond). In some embodiments, the PDZ1 protein can be fused to an envelope protein, which can optionally be binding deficient and/or fusion competent virus envelope protein and included in a lentiviral vector. In some embodiments, the TEFCA can be fused to a cell targeting peptide and the TEFCA-CPT fusion construct can be incorporated into the same or a different lentiviral vector as the PDZ1-envenlope protein construct. During virus production, specific interaction between the PDZ1 and TEFCA facilitates producing virus particles covalently functionalized with the cell targeting peptide and thus capable of targeting a specific cell-type based upon a specific interaction between the cell targeting peptide and cells expressing its binding partner. This approach can be advantageous for use where surface-incompatibilities can restrict the use of, e.g., cell targeting peptides. 
     Lentiviral vectors have been disclosed as in the treatment for Parkinson&#39;s Disease, see, e.g., US Patent Publication No. 20120295960 and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have also been disclosed for the treatment of ocular diseases, see e.g., US Patent Publication Nos. 20060281180, 20090007284, US20110117189; US20090017543; US20070054961, US20100317109. Lentiviral vectors have also been disclosed for delivery to the brain, see, e.g., US Patent Publication Nos. US20110293571; US20110293571, US20040013648, US20070025970, US20090111106 and U.S. Pat. No. 7,259,015. Any of these systems or a variant thereof can be used to deliver a perturbation construct described herein to a cell. 
     In some embodiments, a lentiviral vector system can include one or more transfer plasmids. Transfer plasmids can be generated from various other vector backbones and can include one or more features that can work with other retroviral and/or lentiviral vectors in the system that can, for example, improve safety of the vector and/or vector system, increase virial titers, and/or increase or otherwise enhance expression of the desired insert to be expressed and/or packaged into the viral particle. Suitable features that can be included in a transfer plasmid can include, but are not limited to, 5′LTR, 3′LTR, SIN/LTR, origin of replication (Ori), selectable marker genes (e.g., antibiotic resistance genes), Psi (T), RRE (rev response element), cPPT (central polypurine tract), promoters, WPRE (woodchuck hepatitis post-transcriptional regulatory element), SV40 polyadenylation signal, pUC origin, SV40 origin, F1 origin, and combinations thereof. 
     In another embodiment, Cocal vesiculovirus envelope pseudotyped retroviral or lentiviral vector particles are contemplated (see, e.g., US Patent Publication No. 20120164118 assigned to the Fred Hutchinson Cancer Research Center). Cocal virus is in the Vesiculovirus genus and is a causative agent of vesicular stomatitis in mammals. Cocal virus was originally isolated from mites in Trinidad (Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964)), and infections have been identified in Trinidad, Brazil, and Argentina from insects, cattle, and horses. Many of the vesiculoviruses that infect mammals have been isolated from naturally infected arthropods, suggesting that they are vector-borne. Antibodies to vesiculoviruses are common among people living in rural areas where the viruses are endemic and laboratory-acquired; infections in humans usually result in influenza-like symptoms. The Cocal virus envelope glycoprotein shares 71.5% identity at the amino acid level with VSV-G Indiana, and phylogenetic comparison of the envelope gene of vesiculoviruses shows that Cocal virus is serologically distinct from, but most closely related to, VSV-G Indiana strains among the vesiculoviruses. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) and Travassos da Rosa et al., Am. J. Tropical Med. &amp; Hygiene 33:999-1006 (1984). The Cocal vesiculovirus envelope pseudotyped retroviral vector particles may include for example, lentiviral, alpharetroviral, betaretroviral, gammaretroviral, deltaretroviral, and epsilonretroviral vector particles that may comprise retroviral Gag, Pol, and/or one or more accessory protein(s) and a Cocal vesiculovirus envelope protein. In certain embodiments of these embodiments, the Gag, Pol, and accessory proteins are lentiviral and/or gammaretroviral. In some embodiments, a retroviral vector can contain encoding polypeptides for one or more Cocal vesiculovirus envelope proteins such that the resulting viral or pseudoviral particles are Cocal vesiculovirus envelope pseudotyped. 
     Adenoviral Vectors, Helper-Dependent Adenoviral Vectors, and Hybrid Adenoviral Vectors 
     In some embodiments, the vector can be an adenoviral vector. In some embodiments, the adenoviral vector can include elements such that the virus particle produced using the vector or system thereof can be serotype 2 or serotype 5. In some embodiments, the polynucleotide to be delivered via the adenoviral particle can be up to about 8 kb. Thus, in some embodiments, an adenoviral vector can include a DNA polynucleotide to be delivered that can range in size from about 0.001 kb to about 8 kb. Adenoviral vectors have been used successfully in several contexts (see e.g., Teramato et al. 2000. Lancet. 355:1911-1912; Lai et al. 2002. DNA Cell. Biol. 21:895-913; Flotte et al., 1996. Hum. Gene. Ther. 7:1145-1159; and Kay et al. 2000. Nat. Genet. 24:257-261. 
     In some embodiments the vector can be a helper-dependent adenoviral vector or system thereof. These are also referred to in the art as “gutless” or “gutted” vectors and are a modified generation of adenoviral vectors (see e.g., Thrasher et al. 2006. Nature. 443:E5-7). In certain embodiments of the helper-dependent adenoviral vector system one vector (the helper) can contain all the viral genes required for replication but contains a conditional gene defect in the packaging domain. The second vector of the system can contain only the ends of the viral genome, one or more CRISPR-Cas polynucleotides, and the native packaging recognition signal, which can allow selective packaged release from the cells (see e.g., Cideciyan et al. 2009. N Engl J Med. 361:725-727). Helper-dependent adenoviral vector systems have been successful for gene delivery in several contexts (see e.g., Simonelli et al. 2010. J Am Soc Gene Ther. 18:643-650; Cideciyan et al. 2009. N Engl J Med. 361:725-727; Crane et al. 2012. Gene Ther. 19(4):443-452; Alba et al. 2005. Gene Ther. 12:18-S27; Croyle et al. 2005. Gene Ther. 12:579-587; Amalfitano et al. 1998. J. Virol. 72:926-933; and Morral et al. 1999. PNAS. 96:12816-12821). The techniques and vectors described in these publications can be adapted for inclusion and delivery of the CRISPR-Cas system polynucleotides described herein. In some embodiments, the polynucleotide to be delivered via the viral particle produced from a helper-dependent adenoviral vector or system thereof can be up to about 37 kb. Thus, in some embodiments, an adenoviral vector can include a DNA polynucleotide to be delivered that can range in size from about 0.001 kb to about 37 kb (see e.g., Rosewell et al. 2011. J. Genet. Syndr. Gene Ther. Suppl. 5:001). 
     In some embodiments, the vector is a hybrid-adenoviral vector or system thereof. Hybrid adenoviral vectors are composed of the high transduction efficiency of a gene-deleted adenoviral vector and the long-term genome-integrating potential of adeno-associated, retroviruses, lentivirus, and transposon based-gene transfer. In some embodiments, such hybrid vector systems can result in stable transduction and limited integration site. See e.g., Balague et al. 2000. Blood. 95:820-828; Morral et al. 1998. Hum. Gene Ther. 9:2709-2716; Kubo and Mitani. 2003. J. Virol. 77(5): 2964-2971; Zhang et al. 2013. PloS One. 8(10) e76771; and Cooney et al. 2015. Mol. Ther. 23(4):667-674), whose techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention. In some embodiments, a hybrid-adenoviral vector can include one or more features of a retrovirus and/or an adeno-associated virus. In some embodiments the hybrid-adenoviral vector can include one or more features of a spuma retrovirus or foamy virus (FV). See e.g., Ehrhardt et al. 2007. Mol. Ther. 15:146-156 and Liu et al. 2007. Mol. Ther. 15:1834-1841, whose techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention. Advantages of using one or more features from the FVs in the hybrid-adenoviral vector or system thereof can include the ability of the viral particles produced therefrom to infect a broad range of cells, a large packaging capacity as compared to other retroviruses, and the ability to persist in quiescent (non-dividing) cells. See also e.g., Ehrhardt et al. 2007. Mol. Ther. 156:146-156 and Shuji et al. 2011. Mol. Ther. 19:76-82, whose techniques and vectors described therein can be modified and adapted for use in the perturbation construct of the present invention. 
     Adeno Associated Viral (AAV) Vectors 
     In an embodiment, the vector can be an adeno-associated virus (AAV) vector. See, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94:1351 (1994). Although similar to adenoviral vectors in some of their features, AAVs have some deficiency in their replication and/or pathogenicity and thus can be safer that adenoviral vectors. In some embodiments the AAV can integrate into a specific site on chromosome 19 of a human cell with no observable side effects. In some embodiments, the capacity of the AAV vector, system thereof, and/or AAV particles can be up to about 4.7 kb. The AAV vector or system thereof can include one or more regulatory molecules. In some embodiments the regulatory molecules can be promoters, enhancers, repressors and the like, which are described in greater detail elsewhere herein. In some embodiments, the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory proteins. In some embodiments, the one or more regulatory proteins can be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof. 
     The AAV vector or system thereof can include one or more polynucleotides that can encode one or more capsid proteins. The capsid proteins can be selected from VP1, VP2, VP3, and combinations thereof. The capsid proteins can be capable of assembling into a protein shell of the AAV virus particle. In some embodiments, the AAV capsid can contain 60 capsid proteins. In some embodiments, the ratio of VP1:VP2:VP3 in a capsid can be about 1:1:10. 
     In some embodiments, the AAV vector or system thereof can include one or more adenovirus helper factors or polynucleotides that can encode one or more adenovirus helper factors. Such adenovirus helper factors can include, but are not limited, E1A, E1B, E2A, E4ORF6, and VA RNAs. In some embodiments, a producing host cell line expresses one or more of the adenovirus helper factors. 
     The AAV vector or system thereof can be configured to produce AAV particles having a specific serotype. In some embodiments, the serotype can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9 or any combinations thereof. In some embodiments, the AAV can be AAV1, AAV-2, AAV-5 or any combination thereof. One can select the AAV of the AAV with regard to the cells to be targeted, e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof for targeting brain and/or neuronal cells; and one can select AAV-4 for targeting cardiac tissue; and one can select AAV8 for delivery to the liver. Thus, in some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and/or neuronal cells can be configured to generate AAV particles having serotypes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof. In some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue can be configured to generate an AAV particle having an AAV-4 serotype. In some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver can be configured to generate an AAV having an AAV-8 serotype. In some embodiments, the AAV vector is a hybrid AAV vector or system thereof. Hybrid AAVs are AAVs that include genomes with elements from one serotype that are packaged into a capsid derived from at least one different serotype. For example, if it is the rAAV2/5 that is to be produced, and if the production method is based on the helper-free, transient transfection method discussed above, the 1st plasmid and the 3rd plasmid (the adeno helper plasmid) will be the same as discussed for rAAV2 production. However, the second plasmid, the pRepCap will be different. In this plasmid, called pRep2/Cap5, the Rep gene is still derived from AAV2, while the Cap gene is derived from AAVS. The production scheme is the same as the above-mentioned approach for AAV2 production. The resulting rAAV is called rAAV2/5, in which the genome is based on recombinant AAV2, while the capsid is based on AAVS. It is assumed the cell or tissue-tropism displayed by this AAV2/5 hybrid virus should be the same as that of AAVS. 
     A tabulation of certain AAV serotypes as to these cells can be found in Grimm, D. et al, J. Virol. 82: 5887-5911 (2008). 
     In some embodiments, the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector. In some embodiments, the “gutless” AAV vector or system thereof can have the cis-acting viral DNA elements involved in genome amplification and packaging in linkage with the heterologous sequences of interest (e.g., the perturbation construct (s)). 
     In some embodiments, the AAV vectors are produced in in insect cells, e.g.,  Spodoptera frugiperda  Sf9 insect cells, grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial vendors, e.g., Sigma Aldrich (EX-CELL 405). 
     In some embodiments, an AAV vector or vector system can contain or consists essentially of one or more polynucleotides encoding one or more components of a perturbation construct described herein. In some embodiments, the AAV vector or vector system can contain a plurality of cassettes comprising or consisting a first cassette comprising or consisting essentially of a two or more gRNAs (or their encoding polynucleotides), reporter gene, barcode, and a terminator, advantageously up to the packaging size limit of the vector, e.g., in total. 
     In one embodiment, the invention provides a non-naturally occurring or engineered composition comprising a perturbation construct, which is part of or tethered to an AAV capsid domain, i.e., VP1, VP2, or VP3 domain of Adeno-Associated Virus (AAV) capsid. In some embodiments, part of or tethered to an AAV capsid domain includes associated with associated with an AAV capsid domain. In some embodiments, the perturbation construct may be fused to the AAV capsid domain. In some embodiments, the fusion may be to the N-terminal end of the AAV capsid domain. As such, in some embodiments, the C-terminal end of the CRISPR enzyme is fused to the N-terminal end of the AAV capsid domain. In some embodiments, an NLS and/or a linker (such as a GlySer linker) may be positioned between the C-terminal end of the CRISPR enzyme and the N-terminal end of the AAV capsid domain. In some embodiments, the fusion may be to the C-terminal end of the AAV capsid domain. In some embodiments, this is not preferred due to the fact that the VP1, VP2 and VP3 domains of AAV are alternative splices of the same RNA and so a C-terminal fusion may affect all three domains. In some embodiments, the AAV capsid domain is truncated. In some embodiments, some or all of the AAV capsid domain is removed. In some embodiments, some of the AAV capsid domain is removed and replaced with a linker (such as a GlySer linker), typically leaving the N-terminal and C-terminal ends of the AAV capsid domain intact, such as the first 2, 5 or 10 amino acids. In this way, the internal (non-terminal) portion of the VP3 domain may be replaced with a linker. It is particularly preferred that the linker is fused to the CRISPR protein. A branched linker may be used, with the perturbation construct or component thereof fused to the end of one of the branches. This allows for some degree of spatial separation between the capsid and the perturbation construct or component thereof. In this way, the perturbation construct or component thereof is part of (or fused to) the AAV capsid domain. 
     In other embodiments, the perturbation construct or component thereof may be fused in frame within, i.e., internal to, the AAV capsid domain. Thus, in some embodiments, the AAV capsid domain again preferably retains its N-terminal and C-terminal ends. In this case, a linker is preferred, in some embodiments, either at one or both ends of the perturbation construct. In this way, the perturbation construct or component thereof is again part of (or fused to) the AAV capsid domain. In certain embodiments, the positioning of the perturbation construct or component thereof is such that the perturbation construct or component thereof is at the external surface of the viral capsid once formed. In one embodiment, the invention provides a non-naturally occurring or engineered composition comprising a perturbation construct or component thereof associated with a AAV capsid domain of Adeno-Associated Virus (AAV) capsid. Here, associated may mean in some embodiments fused, or in some embodiments bound to, or in some embodiments tethered to. The perturbation construct or component thereof may, in some embodiments, be tethered to the VP1, VP2, or VP3 domain. This may be via a connector protein or tethering system such as the biotin-streptavidin system. In one example, a biotinylation sequence (15 amino acids) could therefore be fused to the perturbation construct or component thereof. When a fusion of the AAV capsid domain, especially the N-terminus of the AAV AAV capsid domain, with streptavidin is also provided, the two will therefore associate with very high affinity. Thus, in some embodiments, provided is a composition or system comprising a perturbation construct or component thereof—biotin fusion and a streptavidin—AAV capsid domain arrangement, such as a fusion. The perturbation construct or component thereof—biotin and streptavidin—AAV capsid domain forms a single complex when the two parts are brought together. NLSs may also be incorporated between the perturbation construct or component thereof and the biotin; and/or between the streptavidin and the AAV capsid domain. 
     As such, provided is a fusion of a perturbation construct or component thereof with a connector protein specific for a high affinity ligand for that connector, whereas the AAV VP2 domain is bound to said high affinity ligand. For example, streptavidin may be the connector fused to the CRISPR enzyme, while biotin may be bound to the AAV VP2 domain. Upon co-localization, the streptavidin will bind to the biotin, thus connecting the perturbation construct or component thereof to the AAV VP2 domain. The reverse arrangement is also possible. In some embodiments, a biotinylation sequence (15 amino acids) could therefore be fused to the AAV VP2 domain, especially the N-terminus of the AAV VP2 domain. A fusion of the perturbation construct or component thereof with streptavidin is also preferred, in some embodiments. In some embodiments, the biotinylated AAV capsids with streptavidin—perturbation construct or component thereof are assembled in vitro. This way the AAV capsids should assemble in a straightforward manner and the perturbation construct or component thereof—streptavidin fusion can be added after assembly of the capsid. In other embodiments a biotinylation sequence (15 amino acids) could therefore be fused to the perturbation construct or component thereof, together with a fusion of the AAV VP2 domain, especially the N-terminus of the AAV VP2 domain, with streptavidin. For simplicity, a fusion of the perturbation construct or component thereof and the AAV VP2 domain is preferred in some embodiments. In some embodiments, the fusion may be to the N-terminal end of the perturbation construct or component thereof. In other words, in some embodiments, the AAV and perturbation construct or component thereof are associated via fusion. In some embodiments, the AAV and perturbation construct or component thereof are associated via fusion including a linker. Suitable linkers are discussed herein include, but are not limited to, Gly Ser linkers. Fusion to the N-term of AAV VP2 domain is preferred, in some embodiments. In some embodiments, the perturbation construct or component thereof comprises at least one Nuclear Localization Signal (NLS). In a further embodiment, the present invention provides compositions comprising the perturbation construct or component thereof and associated AAV VP2 domain or the polynucleotides or vectors described herein. Such compositions and formulations are discussed elsewhere herein. 
     An alternative tether may be to fuse or otherwise associate the AAV capsid domain to an adaptor protein which binds to or recognizes to a corresponding RNA sequence or motif. In some embodiments, the adaptor is or comprises a binding protein which recognizes and binds (or is bound by) an RNA sequence specific for said binding protein. In some embodiments, a preferred example is the MS2 (see Konermann et al. December 2014, cited infra, incorporated herein by reference) binding protein which recognizes and binds (or is bound by) an RNA sequence specific for the MS2 protein. 
     With the AAV capsid domain associated with the adaptor protein, the perturbation construct or component thereof may, in some embodiments, be tethered to the adaptor protein of the AAV capsid domain. The perturbation construct or component thereof may, in some embodiments, be tethered to the adaptor protein of the AAV capsid domain via the C perturbation construct or component thereof being in a complex with a modified guide, see Konermann et al. The modified guide is, in some embodiments, a sgRNA. In some embodiments, the modified guide comprises a distinct RNA sequence; see, e.g., International Patent Application No. PCT/US14/70175, incorporated herein by reference. 
     In some embodiments, distinct RNA sequence is an aptamer. Thus, corresponding aptamer-adaptor protein systems are preferred. One or more functional domains may also be associated with the adaptor protein. An example of a preferred arrangement would be: [AAV AAV capsid domain-adaptor protein]-[modified guide-perturbation construct or component thereof] 
     In certain embodiments, the positioning of the perturbation construct or component thereof is such that the perturbation construct or component thereof is at the internal surface of the viral capsid once formed. In one embodiment, the invention provides a non-naturally occurring or engineered composition comprising a perturbation construct or component thereof associated with an internal surface of an AAV capsid domain. Here again, associated may mean in some embodiments fused, or in some embodiments bound to, or in some embodiments tethered to. The perturbation construct or component thereof may, in some embodiments, be tethered to the VP1, VP2, or VP3 domain such that it locates to the internal surface of the viral capsid once formed. This may be via a connector protein or tethering system such as the biotin-streptavidin system as described above and/or elsewhere herein. 
     Herpes Simplex Viral Vectors 
     In some embodiments, the vector can be a Herpes Simplex Viral (HSV)-based vector or system thereof. HSV systems can include the disabled infections single copy (DISC) viruses, which are composed of a glycoprotein H defective mutant HSV genome. When the defective HSV is propagated in complementing cells, virus particles can be generated that are capable of infecting subsequent cells permanently replicating their own genome but are not capable of producing more infectious particles. See e.g., 2009. Trobridge. Exp. Opin. Biol. Ther. 9:1427-1436, whose techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention. In some embodiments where an HSV vector or system thereof is utilized, the host cell can be a complementing cell. In some embodiments, HSV vector or system thereof can be capable of producing virus particles capable of delivering a polynucleotide cargo of up to 150 kb. Thus, in some embodiment the CRISPR-Cas system polynucleotide(s) included in the HSV-based viral vector or system thereof can sum from about 0.001 to about 150 kb. HSV-based vectors and systems thereof have been successfully used in several contexts including various models of neurologic disorders. See e.g., Cockrell et al. 2007. Mol. Biotechnol. 36:184-204; Kafri T. 2004. Mol. Biol. 246:367-390; Balaggan and Ali. 2012. Gene Ther. 19:145-153; Wong et al. 2006. Hum. Gen. Ther. 2002. 17:1-9; Azzouz et al. J. Neruosci. 22L10302-10312; and Betchen and Kaplitt. 2003. Curr. Opin. Neurol. 16:487-493, whose techniques and vectors described therein can be modified and adapted for use in the CRISPR-Cas system of the present invention. 
     Poxvirus Vectors 
     In some embodiments, the vector can be a poxvirus vector or system thereof. In some embodiments, the poxvirus vector can result in cytoplasmic expression of perturbation construct or component thereof the present invention. In some embodiments the capacity of a poxvirus vector or system thereof can be about 25 kb or more. In some embodiments, a poxvirus vector or system thereof can include one or more perturbation constructs or component thereof described herein. 
     Virus Particle Production from Viral Vectors 
     Retroviral Production 
     In some embodiments, one or more viral vectors and/or system thereof can be delivered to a suitable cell line for production of virus particles containing the polynucleotide or other payload to be delivered to a host cell. Suitable host cells for virus production from viral vectors and systems thereof described herein are known in the art and are commercially available. For example, suitable host cells include HEK 293 cells and its variants (HEK 293T and HEK 293TN cells). In some embodiments, the suitable host cell for virus production from viral vectors and systems thereof described herein can stably express one or more genes involved in packaging (e.g., pol, gag, and/or VSV-G) and/or other supporting genes. 
     In some embodiments, after delivery of one or more viral vectors to the suitable host cells for or virus production from viral vectors and systems thereof, the cells are incubated for an appropriate length of time to allow for viral gene expression from the vectors, packaging of the polynucleotide to be delivered (e.g., a perturbation construct or component thereof), and virus particle assembly, and secretion of mature virus particles into the culture media. Various other methods and techniques are generally known to those of ordinary skill in the art. 
     Mature virus particles can be collected from the culture media by a suitable method. In some embodiments, this can involve centrifugation to concentrate the virus. The titer of the composition containing the collected virus particles can be obtained using a suitable method. Such methods can include transducing a suitable cell line (e.g., NIH 3T3 cells) and determining transduction efficiency, infectivity in that cell line by a suitable method. Suitable methods include PCR-based methods, flow cytometry, and antibiotic selection-based methods. Various other methods and techniques are generally known to those of ordinary skill in the art. The concentration of virus particle can be adjusted as needed. In some embodiments, the resulting composition containing virus particles can contain 1×10 1 -1×10 20  particles/mL. 
     Lentiviruses may be prepared from any lentiviral vector or vector system described herein. In one example embodiment, after cloning pCasES10 (which contains a lentiviral transfer plasmid backbone), HEK293FT at low passage (p=5) can be seeded in a T-75 flask to 50% confluence the day before transfection in DMEM with 10% fetal bovine serum and without antibiotics. After 20 hours, the media can be changed to OptiMEM (serum-free) media and transfection of the lentiviral vectors can done 4 hours later. Cells can be transfected with 10 μg of lentiviral transfer plasmid (pCasES10) and the appropriate packaging plasmids (e.g., 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 ug of psPAX2 (gag/pol/rev/tat)). Transfection can be carried out in 4 mL OptiMEM with a cationic lipid delivery agent (50 uL Lipofectamine 2000 and 100 ul Plus reagent). After 6 hours, the media can be changed to antibiotic-free DMEM with 10% fetal bovine serum. These methods can use serum during cell culture, but serum-free methods are preferred. 
     Following transfection and allowing the producing cells (also referred to as packaging cells) to package and produce virus particles with packaged cargo, the lentiviral particles can be purified. In an exemplary embodiment, virus-containing supernatants can be harvested after 48 hours. Collected virus-containing supernatants can first be cleared of debris and filtered through a 0.45 um low protein binding (PVDF) filter. They can then be spun in an ultracentrifuge for 2 hours at 24,000 rpm. The resulting virus-containing pellets can be resuspended in 50 ul of DMEM overnight at 4 degrees C. They can be then aliquoted and used immediately or immediately frozen at −80 degrees C. for storage. 
     Pooling of Virus Particles 
     In some embodiments, the virus particles (e.g., lentiviral particles) containing the gRNAs, the reporter gene, and the barcode is packaged individually for each perturbation construct and/or each target gene. In some embodiments, the lentiviruses containing the gRNAs, the reporter gene, and the barcode for multiple targets are packaged in a pool or in an array manner. 
     In some embodiments, the virus particles (e.g., lentiviral particles containing gRNAs for each target gene are pooled with equal titer to minimize vector recombination so that each individual type of lentiviruses that representing each individual type of gRNA for a target gene has an equal representation in the pool. 
     In some embodiments, the pool of lentiviruses is delivered by injection into an anatomic site or anatomic sites in vivo that contain the desired progenitor cells. As a result, one or more progenitor cells are transduced with the viruses delivered, and the gRNAs together with reporter(s) and barcode are expressed in the transduced progenitor cells. 
     AAV Particle Production 
     There are two main strategies for producing AAV particles from AAV vectors and systems thereof, such as those described herein, which depend on how the adenovirus helper factors are provided (helper v. helper free). In some embodiments, a method of producing AAV particles from AAV vectors and systems thereof can include adenovirus infection into cell lines that stably harbor AAV replication and capsid encoding polynucleotides along with AAV vector containing the polynucleotide to be packaged and delivered by the resulting AAV particle (e.g., the perturbation construct or component thereof (s)). In some embodiments, a method of producing AAV particles from AAV vectors and systems thereof can be a “helper free” method, which includes co-transfection of an appropriate producing cell line with three vectors (e.g., plasmid vectors): (1) an AAV vector that contains a polynucleotide of interest (e.g., the perturbation construct or component thereof (s)) between 2 ITRs; (2) a vector that carries the AAV Rep-Cap encoding polynucleotides; and (helper polynucleotides. One of skill in the art will appreciate various methods and variations thereof that are both helper and—helper free and as well as the different advantages of each system. 
     Barcodes 
     As described elsewhere herein the perturbation construct includes a barcode that can be operably linked to the reporter gene and gRNAs. In certain example embodiments, the barcode is polyadenylated. 
     The term “barcode” as used herein refers to a short sequence of nucleotides (for example, DNA or RNA) that is used as an identifier for an associated molecule, such as a target molecule and/or target nucleic acid, or as an identifier of the source of an associated molecule, such as a cell-of-origin. A barcode may also refer to any unique, non-naturally occurring, nucleic acid sequence that may be used to identify the originating source of a nucleic acid fragment. Although it is not necessary to understand the mechanism of an invention, it is believed that the barcode sequence provides a high-quality individual read of a barcode associated with a single cell, a viral vector, labeling ligand (e.g., an aptamer), protein, shRNA, sgRNA or cDNA such that multiple species can be sequenced together. 
     Barcoding may be performed based on any of the compositions or methods disclosed in patent publication WO 2014047561 A1, Compositions and methods for labeling of agents, incorporated herein in its entirety. In certain embodiments barcoding uses an error correcting scheme (T. K. Moon, Error Correction Coding: Mathematical Methods and Algorithms (Wiley, New York, ed. 1, 2005)). Not being bound by a theory, amplified sequences from single cells can be sequenced together and resolved based on the barcode associated with each cell. 
     In preferred embodiments, sequencing is performed using unique molecular identifiers (UMI). The term “unique molecular identifiers” (UMI) as used herein refers to a sequencing linker or a subtype of nucleic acid barcode used in a method that uses molecular tags to detect and quantify unique amplified products. A UMI is used to distinguish effects through a single clone from multiple clones. The term “clone” as used herein may refer to a single mRNA or target nucleic acid to be sequenced. The UMI may also be used to determine the number of transcripts that gave rise to an amplified product, or in the case of target barcodes as described herein, the number of binding events. In preferred embodiments, the amplification is by PCR or multiple displacement amplification (MDA). 
     In certain embodiments, an UMI with a random sequence of between 4 and 20 base pairs is added to a template, which is amplified and sequenced. In preferred embodiments, the UMI is added to the 5′ end of the template. Sequencing allows for high resolution reads, enabling accurate detection of true variants. As used herein, a “true variant” will be present in every amplified product originating from the original clone as identified by aligning all products with a UMI. Each clone amplified will have a different random UMI that will indicate that the amplified product originated from that clone. Background caused by the fidelity of the amplification process can be eliminated because true variants will be present in all amplified products and background representing random error will only be present in single amplification products (See e.g., Islam S. et al., 2014. Nature Methods No: 11, 163-166). Not being bound by a theory, the UMI&#39;s are designed such that assignment to the original can take place despite up to 4-7 errors during amplification or sequencing. Not being bound by a theory, an UMI may be used to discriminate between true barcode sequences. 
     Unique molecular identifiers can be used, for example, to normalize samples for variable amplification efficiency. For example, in various embodiments, featuring a solid or semisolid support (for example a hydrogel bead), to which nucleic acid barcodes (for example a plurality of barcodes sharing the same sequence) are attached, each of the barcodes may be further coupled to a unique molecular identifier, such that every barcode on the particular solid or semisolid support receives a distinct unique molecule identifier. A unique molecular identifier can then be, for example, transferred to a target molecule with the associated barcode, such that the target molecule receives not only a nucleic acid barcode, but also an identifier unique among the identifiers originating from that solid or semisolid support. 
     A nucleic acid barcode or UMI can have a length of at least, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides, and can be in single- or double-stranded form. Target molecule and/or target nucleic acids can be labeled with multiple nucleic acid barcodes in combinatorial fashion, such as a nucleic acid barcode concatemer. Typically, a nucleic acid barcode is used to identify a target molecule and/or target nucleic acid as being from a particular discrete volume, having a particular physical property (for example, affinity, length, sequence, etc.), or having been subject to certain treatment conditions. Target molecule and/or target nucleic acid can be associated with multiple nucleic acid barcodes to provide information about all of these features (and more). Each member of a given population of UMIs, on the other hand, is typically associated with (for example, covalently bound to or a component of the same molecule as) individual members of a particular set of identical, specific (for example, discreet volume-, physical property-, or treatment condition-specific) nucleic acid barcodes. Thus, for example, each member of a set of origin-specific nucleic acid barcodes, or other nucleic acid identifier or connector oligonucleotide, having identical or matched barcode sequences, may be associated with (for example, covalently bound to or a component of the same molecule as) a distinct or different UMI. 
     As disclosed herein, unique nucleic acid identifiers are used to label the target molecules and/or target nucleic acids, for example origin-specific barcodes and the like. The nucleic acid identifiers, nucleic acid barcodes, can include a short sequence of nucleotides that can be used as an identifier for an associated molecule, location, or condition. In certain embodiments, the nucleic acid identifier further includes one or more unique molecular identifiers and/or barcode receiving adapters. A nucleic acid identifier can have a length of about, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 base pairs (bp) or nucleotides (nt). In certain embodiments, a nucleic acid identifier can be constructed in combinatorial fashion by combining randomly selected indices (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 indexes). Each such index is a short sequence of nucleotides (for example, DNA, RNA, or a combination thereof) having a distinct sequence. An index can have a length of about, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bp or nt. Nucleic acid identifiers can be generated, for example, by split-pool synthesis methods, such as those described, for example, in International Patent Publication Nos. WO 2014/047556 and WO 2014/143158, each of which is incorporated by reference herein in its entirety. 
     In some embodiments, the expression of reporter gene and barcode is controlled by a separate promoter from those used for controlling the expression of gRNAs. Configuration of the barcode within the perturbation construct is described in greater detail elsewhere herein. 
     Barcode with Cleavage Sites 
     A nucleic acid barcode may be cleavable from a specific binding agent, for example, after the specific binding agent has bound to a target molecule. In some embodiments, the origin-specific barcode further comprises one or more cleavage sites. In some examples, at least one cleavage site is oriented such that cleavage at that site releases the origin-specific barcode from a substrate, such as a bead, for example a hydrogel bead, to which it is coupled. In some examples, at least one cleavage site is oriented such that the cleavage at the site releases the origin-specific barcode from the target molecule specific binding agent. In some examples, a cleavage site is an enzymatic cleavage site, such an endonuclease site present in a specific nucleic acid sequence. In other embodiments, a cleavage site is a peptide cleavage site, such that a particular enzyme can cleave the amino acid sequence. In still other embodiments, a cleavage site is a site of chemical cleavage. 
     Barcode Adapters 
     In some embodiments, the target molecule is attached to an origin-specific barcode receiving adapter, such as a nucleic acid. In some examples, the origin-specific barcode receiving adapter comprises an overhang and the origin-specific barcode comprises a sequence capable of hybridizing to the overhang. A barcode receiving adapter is a molecule configured to accept or receive a nucleic acid barcode, such as an origin-specific nucleic acid barcode. For example, a barcode receiving adapter can include a single-stranded nucleic acid sequence (for example, an overhang) capable of hybridizing to a given barcode (for example, an origin-specific barcode), for example, via a sequence complementary to a portion or the entirety of the nucleic acid barcode. In certain embodiments, this portion of the barcode is a standard sequence held constant between individual barcodes. The hybridization couples the barcode receiving adapter to the barcode. In some embodiments, the barcode receiving adapter may be associated with (for example, attached to) a target molecule. As such, the barcode receiving adapter may serve as the means through which an origin-specific barcode is attached to a target molecule. A barcode receiving adapter can be attached to a target molecule according to methods known in the art. For example, a barcode receiving adapter can be attached to a polypeptide target molecule at a cysteine residue (for example, a C-terminal cysteine residue). A barcode receiving adapter can be used to identify a particular condition related to one or more target molecules, such as a cell of origin or a discreet volume of origin. For example, a target molecule can be a cell surface protein expressed by a cell, which receives a cell-specific barcode receiving adapter. The barcode receiving adapter can be conjugated to one or more barcodes as the cell is exposed to one or more conditions, such that the original cell of origin for the target molecule, as well as each condition to which the cell was exposed, can be subsequently determined by identifying the sequence of the barcode receiving adapter/barcode concatemer. 
     Barcode with Capture Moiety 
     In some embodiments, an origin-specific barcode further includes a capture moiety, covalently or non-covalently linked. Thus, in some embodiments the origin-specific barcode, and anything bound or attached thereto, that include a capture moiety are captured with a specific binding agent that specifically binds the capture moiety. In some embodiments, the capture moiety is adsorbed or otherwise captured on a surface. In specific embodiments, a targeting probe is labeled with biotin, for instance by incorporation of biotin-16-UTP during in vitro transcription, allowing later capture by streptavidin. Other means for labeling, capturing, and detecting an origin-specific barcode include incorporation of aminoallyl-labeled nucleotides, incorporation of sulfhydryl-labeled nucleotides, incorporation of allyl- or azide-containing nucleotides, and many other methods described in Bioconjugate Techniques (2nd Ed), Greg T. Hermanson, Elsevier (2008), which is specifically incorporated herein by reference. In some embodiments, the targeting probes are covalently coupled to a solid support or other capture device prior to contacting the sample, using methods such as incorporation of aminoallyl-labeled nucleotides followed by 1-Ethyl-3-β-dimethylaminopropyl)carbodiimide (EDC) coupling to a carboxy-activated solid support, or other methods described in Bioconjugate Techniques. In some embodiments, the specific binding agent has been immobilized for example on a solid support, thereby isolating the origin-specific barcode. 
     Other Barcoding Embodiments 
     DNA barcoding is also a taxonomic method that uses a short genetic marker in an organism&#39;s DNA to identify it as belonging to a particular species. It differs from molecular phylogeny in that the main goal is not to determine classification but to identify an unknown sample in terms of a known classification. Kress et al., “Use of DNA barcodes to identify flowering plants” Proc. Natl. Acad. Sci. U.S.A. 102(23):8369-8374 (2005). Barcodes are sometimes used in an effort to identify unknown species or assess whether species should be combined or separated. Koch H., “Combining morphology and DNA barcoding resolves the taxonomy of Western Malagasy Liotrigona Moure, 1961” African Invertebrates 51(2): 413-421 (2010); and Seberg et al., “How many loci does it take to DNA barcode a  crocus ?” PLoS One 4(2):e4598 (2009). Barcoding has been used, for example, for identifying plant leaves even when flowers or fruit are not available, identifying the diet of an animal based on stomach contents or feces, and/or identifying products in commerce (for example, herbal supplements or wood). Soininen et al., “Analysing diet of small herbivores: the efficiency of DNA barcoding coupled with high-throughput pyrosequencing for deciphering the composition of complex plant mixtures” Frontiers in Zoology 6:16 (2009). 
     It has been suggested that a desirable locus for DNA barcoding should be standardized so that large databases of sequences for that locus can be developed. Most of the taxa of interest have loci that are sequencable without species-specific PCR primers. CBOL Plant Working Group, “A DNA barcode for land plants” PNAS 106(31):12794-12797 (2009). Further, these putative barcode loci are believed short enough to be easily sequenced with current technology. Kress et al., “DNA barcodes: Genes, genomics, and bioinformatics” PNAS 105(8):2761-2762 (2008). Consequently, these loci would provide a large variation between species in combination with a relatively small amount of variation within a species. Lahaye et al., “DNA barcoding the floras of biodiversity hotspots” Proc Natl Acad Sci USA 105(8):2923-2928 (2008). 
     DNA barcoding is based on a relatively simple concept. For example, most eukaryote cells contain mitochondria, and mitochondrial DNA (mtDNA) has a relatively fast mutation rate, which results in significant variation in mtDNA sequences between species and, in principle, a comparatively small variance within species. A 648-bp region of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene was proposed as a potential ‘barcode’. As of 2009, databases of CO1 sequences included at least 620,000 specimens from over 58,000 species of animals, larger than databases available for any other gene. Ausubel, J., “A botanical macroscope” Proceedings of the National Academy of Sciences 106(31):12569 (2009). 
     Software for DNA barcoding requires integration of a field information management system (FIMS), laboratory information management system (LIMS), sequence analysis tools, workflow tracking to connect field data and laboratory data, database submission tools and pipeline automation for scaling up to eco-system scale projects. Geneious Pro can be used for the sequence analysis components, and the two plugins made freely available through the Moorea Biocode Project, the Biocode LIMS and Genbank Submission plugins handle integration with the FIMS, the LIMS, workflow tracking and database submission. 
     Additionally, other barcoding designs and tools have been described (see e.g., Birrell et al., (2001) Proc. Natl Acad. Sci. USA 98, 12608-12613; Giaever, et al., (2002) Nature 418, 387-391; Winzeler et al., (1999) Science 285, 901-906; and Xu et al., (2009) Proc Natl Acad Sci USA. February 17; 106(7):2289-94). 
     Delivery of a Perturbation Construct to Animal Model Cells 
     In some embodiments, the method includes introducing a plurality of genetic perturbations in a plurality of cells in an animal model, such as a Cas animal model. In some embodiments, introduction of a plurality of genetic perturbations includes delivering a pool of engineered virus particles to the animal model such that one or more of the cells in the animal model are transduced. In some embodiments, introducing a plurality of genetic perturbations in a plurality of cells in an animal model occurs at one or more timepoints during embryonic development. In some embodiments, introducing a plurality of genetic perturbations occurs at one or more time points post-partum. In some embodiments, introducing a plurality of genetic perturbations is induced by triggering an inducible promoter of the perturbation construct and/or Cas protein in the Cas animal model such that the gRNAs and/or Cas protein are expressed at the same time. This can allow for both spatial and temporal control over the perturbations. In some embodiments, introduction is cell or tissue specific, which can be controlled by various methods such as spatially controlling delivery a pool of engineered virus particles only to a specific cell or cell population, tissue or other spatial region. 
     In some embodiments, a delivery is to the heart, kidney, lung, skin, pancreas, intestine, bone, bone marrow, fat, spleen, bursa of Fabricius, bladder, blood, placenta, thymus, brain or other central nervous system cell, peripheral nervous system cell, liver, muscle, any other organ, soft tissue, or any combination thereof. 
     In some embodiments, delivery is to one or more progenitor cells. As used herein, “progenitor cell” refers to cells that are early descendants of stem cells that are capable of a limited number of cell divisions and are capable of differentiating to form one or more types of cells. In some embodiment, the progenitor cells are neural progenitor cells, myeloid progenitor cells, multipotent progenitor cells, and/or hematopoietic progenitor cells. It will be appreciated that there is overlap between multi-potent stem cells and progenitor cells. As used herein, “neural progenitor” refers to a progenitor cell of the central nervous system (CNS) that give rise to many, if not all, of the glial and neuronal cell types that populate the CNS (see e.g., Martinez-Cerdeno and Noctor. Front. Neuroanat., 6 Dec. 2018|https://doi.org/10.3389/fnana.2018.00104). 
     In some embodiments, delivery is to a progenitor cell such that progeny carry the perturbation as the progenitor cell divides and/or differentiates. In some embodiments, subsequent steps of the methods described herein (such as enrichment and/or sc-RNA seq) occur after division and/or differentiation of a transduced and/or perturbed cell. 
     In some embodiments, the progenitor cells infected with the lentiviruses develop into a plurality of distinct types of progeny cells. In some embodiments, the neural progenitor cells infected with the lentiviruses develop into a plurality of distinct types of progeny cells. In some embodiments, the progeny cells arise from the lentivirus-infected neural progenitor cells include, but are not limited, projection neurons, interneurons, astroglia, and oligodendrocytes. In some embodiments, the neural progeny cells are located in diverse brain regions. 
     In some embodiments, the progeny cells are collected from the targeted tissue or tissues in the newborn mouse at any time of P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, or after P10. In some embodiments, the progeny cells are collected from the targeted tissue or tissues in the newborn mouse on P7. 
     In some embodiments, the rate of frameshifted insertion/deletion for each gRNA target among the lentiviral infected cells is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. 
     In certain example embodiments, introducing further comprises delivering to the plurality of progenitor cells a pool of engineered virus particles comprising equal genetic perturbation representation. 
     In certain example embodiments, the engineered virus particles are engineered lentiviral particles. 
     In certain example embodiments, introducing further comprises delivering the pool of engineered virus particles to a target tissue of a developing embryo of the Cas animal model in utero. 
     In certain example embodiments, the developing embryo is at stage between E5-E17 or an equivalent stage thereof, such as E5, E6, E7, E8, E9, E10, E11, E12, E13, E14, E15, E16, or E17 or an equivalent stage thereof. In some embodiments, the developing embryo is at stage 12.5 or equivalent thereof. 
     In some embodiments, the lentiviruses are injected into the lateral ventricular zone in a developing embryo in utero at a stage of E5, E6, E7, E8, E9, E10, E11, E12, E13, E14, E15, E16, or E17. In some aspect, the lentiviruses are injected at a stage of E12.5. 
     Enrichment of Perturbed Cells 
     In embodiments, the method includes generating and enriched perturbed and/or reporter gene expressing cell population. As previously discussed, a reporter gene is operably linked and to at least the two or more gRNAs of the perturbation construct. Thus, by identifying, separating and/or isolating the cells expressing the reporter, the result is also an enrichment of perturbed cells. In certain example embodiments, the enriched perturbed cell population comprises progenitor cell progeny. 
     In some embodiments, identification and separation of reporter expressing cells includes FACS. FACS can be performed directly, such as by detecting expression of an optically active reporter, or indirectly by using one or more immunological detection methods to apply an optically active label to reporter expressing cells and performing FACS based on detection of the optically active label. Other methods of detecting, separating, isolating, and thus enriching live cells based on detection of expression of a reporter gene are known and can be used to enrich the population of perturbed cells. 
     In some embodiments, enrichment includes dissecting out a tissue or cell population prior to or alternative to FACS or other separation/isolation method. In some embodiments, dissecting includes microdissection. In some embodiments, transduced progeny cells can be dissected out. In some embodiments, the cell survival rate after FACS is at least 40%, at least 50%, at least 60%, at lease 70%, at least 80%, or at least 90%. 
     Analysis of the Enriched Perturbed Cell Population 
     The method includes phenotypic evaluation or a proxy therefor of the perturbed cells. Such analysis includes the identification of cell types within the enriched perturbed population and determination of gene modules that covary within a cell type. In some embodiments, scRNA-seq is used to identify cell types within the enriched perturbed population. In some embodiments, additional phenotypic analyses are performed. 
     ScRNA-seq 
     Generally, and as previously described, the gene signatures and gene modules are screened by perturbation of target genes within said signatures and modules. Methods and tools for genome-scale screening of perturbations in single cells using CRISPR-Cas9 have been described, and are generally referred to as perturb-seq (see e.g., Dixit et al., “Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens” 2016, Cell 167, 1853-1866; Adamson et al., “A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response” 2016, Cell 167, 1867-1882; Feldman et al., Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens, bioRxiv 262121, doi: doi.org/10.1101/262121; Datlinger, et al., 2017, Pooled CRISPR screening with single-cell transcriptome readout. Nature Methods. Vol. 14 No. 3 DOI: 10.1038/nmeth.4177; Hill et al., On the design of CRISPR-based single cell molecular screens, Nat Methods. 2018 April; 15(4): 271-274; Replogle, et al., “Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing” Nat Biotechnol (2020). doi.org/10.1038/s41587-020-0470-y; and International publication serial number WO/2017/075294). It will be appreciated as discussed elsewhere herein that the present disclosure relates to such methods but differs as discussed elsewhere herein. The present invention is compatible with perturb-seq, such that signature genes may be perturbed, and the perturbation may be identified and assigned to the proteomic and gene expression readouts of single cells and can be capable of doing so with greater efficiency. In certain embodiments, a plurality of target genes may be perturbed in single cells and gene expression analyzed. Not being bound by a theory, networks of genes that are disrupted due to perturbation of a signature gene may be determined. Understanding the network of genes effected by a perturbation may allow for a gene to be linked to a specific pathway that may be targeted to modulate the signature and treat a cancer. Thus, in certain embodiments, perturb-seq is used to discover novel gene and drug targets to allow treatment of various diseases in which the target genes are involved. 
     The perturbation methods and tools allow reconstructing of a cellular network or circuit. In one embodiment, the method comprises (1) introducing combinatorial perturbations to a population of cells, (2) measuring genomic, genetic, proteomic, epigenetic and/or phenotypic differences in single cells and (3) assigning a perturbation(s) to the single cells. Not being bound by a theory, a perturbation may be linked to a phenotypic change, preferably changes in gene or protein expression. In preferred embodiments, measured differences that are relevant to the perturbations are determined by applying a model accounting for co-variates to the measured differences. The model may include the capture rate of measured signals, whether the perturbation actually perturbed the cell (phenotypic impact), the presence of subpopulations of either different cells or cell states, and/or analysis of matched cells without any perturbation. In certain embodiments, the measuring of phenotypic differences and assigning a perturbation to a single cell is determined by performing single cell RNA sequencing (RNA-seq). In preferred embodiments, the single cell RNA-seq is performed by any method as described herein (e.g., Drop-seq, InDrop, 10× genomics). In certain embodiments, unique barcodes are used to perform Perturb-seq. In certain embodiments, a guide RNA is detected by RNA-seq using a transcript expressed from a vector encoding the guide RNA. The transcript may include a unique barcode specific to the guide RNA. Not being bound by a theory, a guide RNA and guide RNA barcode is expressed from the same vector and the barcode may be detected by RNA-seq. Not being bound by a theory, detection of a guide RNA barcode is more reliable than detecting a guide RNA sequence, reduces the chance of false guide RNA assignment and reduces the sequencing cost associated with executing these screens. Thus, a perturbation may be assigned to a single cell by detection of a guide RNA barcode in the cell. In certain embodiments, a cell barcode is added to the RNA in single cells, such that the RNA may be assigned to a single cell. Generating cell barcodes is described herein for single cell sequencing methods. In certain embodiments, a Unique Molecular Identifier (UMI) is added to each individual transcript and protein capture oligonucleotide. Not being bound by a theory, the UMI allows for determining the capture rate of measured signals, or preferably the binding events or the number of transcripts captured. Not being bound by a theory, the data is more significant if the signal observed is derived from more than one protein binding event or transcript. In preferred embodiments, Perturb-seq is performed using a guide RNA barcode expressed as a polyadenylated transcript, a cell barcode, and a UMI. 
     In some embodiments, the method includes identifying cell types and corresponding perturbations via single cell RNA sequencing of the enriched perturbed cell population (see, e.g., Kalisky, T., Blainey, P. &amp; Quake, S. R. Genomic Analysis at the Single-Cell Level. Annual review of genetics 45, 431-445, (2011); Kalisky, T. &amp; Quake, S. R. Single-cell genomics. Nature Methods 8, 311-314 (2011); Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Research, (2011); Tang, F. et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nature Protocols 5, 516-535, (2010); Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nature Methods 6, 377-382, (2009); Ramskold, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nature Biotechnology 30, 777-782, (2012); and Hashimshony, T., Wagner, F., Sher, N. &amp; Yanai, I. CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification. Cell Reports, Cell Reports, Volume 2, Issue 3, p 666-673, 2012). 
     In certain embodiments, the invention involves plate based single cell RNA sequencing (see, e.g., Picelli, S. et al., 2014, “Full-length RNA-seq from single cells using Smart-seq2” Nature protocols 9, 171-181, doi:10.1038/nprot.2014.006). 
     In certain embodiments, the invention involves high-throughput single-cell RNA-seq. In this regard reference is made to Macosko et al., 2015, “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets” Cell 161, 1202-1214; International patent application number PCT/US2015/049178, published as WO2016/040476 on Mar. 17, 2016; Klein et al., 2015, “Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell 161, 1187-1201; International patent application number PCT/US2016/027734, published as WO2016168584A1 on Oct. 20, 2016; Zheng, et al., 2016, “Haplotyping germline and cancer genomes with high-throughput linked-read sequencing” Nature Biotechnology 34, 303-311; Zheng, et al., 2017, “Massively parallel digital transcriptional profiling of single cells” Nat. Commun. 8, 14049 doi: 10.1038/ncomms14049; International patent publication number WO2014210353A2; Zilionis, et al., 2017, “Single-cell barcoding and sequencing using droplet microfluidics” Nat Protoc. January; 12(1):44-73; Cao et al., 2017, “Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing” bioRxiv preprint first posted online Feb. 2, 2017, doi: dx.doi.org/10.1101/104844; Rosenberg et al., 2017, “Scaling single cell transcriptomics through split pool barcoding” bioRxiv preprint first posted online Feb. 2, 2017, doi: dx.doi.org/10.1101/105163; Rosenberg et al., “Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding” Science 15 Mar. 2018; Vitak, et al., “Sequencing thousands of single-cell genomes with combinatorial indexing” Nature Methods, 14(3):302-308, 2017; Cao, et al., Comprehensive single-cell transcriptional profiling of a multicellular organism. Science, 357(6352):661-667, 2017; Gierahn et al., “Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput” Nature Methods 14, 395-398 (2017); and Hughes, et al., “Highly Efficient, Massively-Parallel Single-Cell RNA-Seq Reveals Cellular States and Molecular Features of Human Skin Pathology” bioRxiv 689273; doi: doi.org/10.1101/689273, all the contents and disclosure of each of which are herein incorporated by reference in their entirety. 
     In certain embodiments, the invention involves single nucleus RNA sequencing. In this regard reference is made to Swiech et al., 2014, “In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9” Nature Biotechnology Vol. 33, pp. 102-106; Habib et al., 2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons” Science, Vol. 353, Issue 6302, pp. 925-928; Habib et al., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq” Nat Methods. 2017 October; 14(10):955-958; International patent application number PCT/US2016/059239, published as WO2017164936 on Sep. 28, 2017; International patent application number PCT/US2018/060860, published as WO/2019/094984 on May 16, 2019; International patent application number PCT/US2019/055894, published as WO/2020/077236 on Apr. 16, 2020; and Drokhlyansky, et al., “The enteric nervous system of the human and mouse colon at a single-cell resolution,” bioRxiv 746743; doi: doi.org/10.1101/746743, which are herein incorporated by reference in their entirety. 
     In certain embodiments, the invention involves the Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq) as described. (see, e.g., Buenrostro, et al., Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods 2013; 10 (12): 1213-1218; Buenrostro et al., Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486-490 (2015); Cusanovich, D. A., Daza, R., Adey, A., Pliner, H., Christiansen, L., Gunderson, K. L., Steemers, F. J., Trapnell, C. &amp; Shendure, J. Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing. Science. 2015 May 22; 348(6237):910-4. doi: 10.1126/science.aab1601. Epub 2015 May 7; US20160208323A1; US20160060691A1; and WO2017156336A1). 
     In some embodiments, the scRNA-Seq produces a transcriptome of the different types of progeny cells. In some embodiments, the scRNA-Seq produces a gene program or module information of the different types of progeny cells. In some embodiments, the scRNA-Seq produces information about cell states for the progeny cells. 
     Additional Phenotypic Analyses 
     In some embodiments additional analyses are performed. In some embodiments, the enriched progeny cells are subjected to proteomic analysis, genomic analysis, phenotypic analysis, and/or any other relevant biological analyses. In some embodiments, the tissues that contain the progeny cells are subjected to immunohistochemistry analysis to show the expression of proteins. 
     Identification of Gene Modules that covary in a cell type 
     As used herein, a “gene module” is defined as a set of genes within each cell type that co-varied as a group across most cells within a given cell-type cluster. Within each module, the expression of the group of genes is highly correlated with one another. In some embodiments, the modules are used to reflect common biological processes. These common biological processes can be cell cycle, cell differentiation, cell identity, cell death, apoptosis, or any other biological cellular events. 
     In some embodiments, a gene module can be established using a variety of algorithms. In some embodiments, a module can be established using Weighted Gene Correlation Network Analysis (WGCNA). In some other embodiments, a module can be established using Structural Topic Modeling (STM). In some embodiments, a module can be established using other algorithms. 
     In some embodiments, the modules selected using WGCNA is highly correlated with those selected using STM. In some embodiments, modules selected using either WGCNA or STM can be used for subsequent analysis. 
     In some embodiments, a number of WGCNA modules can be used for subsequent analysis. The number of modules can be 1, 2, 3, more than 3, more than 10, more than 15, or more than 50. 
     In some embodiments, a number of modules are extracted from all of relevant cell types. In some embodiments, a number of modules are extracted from major cell types. 
     In some embodiments, some modules are specific to one subcluster within a cell type. In some embodiments, some modules are across cells in multiple subclusters. 
     In some embodiments, the modules are used for testing the association with the perturbation of genes under interrogation. In some embodiments, a linear model is developed to estimate the effect size of each genetic perturbation on that module. As such, the function of each gene perturbed in each cell type can be evaluated using the modules selected. 
     Focusing on gene modules as opposed to individual genes can provide more statistical power to detect biologically meaningful perturbation effects while using fewer cells. In some embodiments, the method includes a determination of gene modules that covary with cell type and/or state. 
     In some embodiments, gene expression modules are generated using WGCNA or STP algorithms. In some embodiments, the modules selected using WGCNA is highly correlated with those selected using STM. In some embodiments, modules selected using either WGCNA or STM can be used for subsequent analysis. 
     In some embodiments, a number of WGCNA modules can be used for subsequent analysis. The number of modules can be 1, 2, 3, more than 3, more than 10, more than 15, or more than 50. 
     In some embodiments, a number of modules are extracted from all of relevant neural cell types. In some embodiments, a number of modules are extracted from major neural cell types. 
     In some embodiments, some modules are specific to one subcluster within a cell type. In some embodiments, some modules are across cells in multiple subclusters. 
     In some embodiments, the modules are used for testing the association with the perturbation of ASD risk-associated genes under interrogation. In some embodiments, a linear model is developed to estimate the effect size of each genetic perturbation of the ASD risk-associated genes on that module. As such, the effect of each ASD risk-associated gene perturbed in each progeny cell type can be evaluated using the modules selected. 
     In Vivo Screening for Therapeutic Targets and Therapeutic Agents 
     In some embodiments, methods for identifying therapeutic targets are disclosed. The targets identified using these methods represent faithfully authentic changes in molecular machinery and the cell states induced the disease. 
     In some embodiments, candidate genes for therapeutic targets can be selected from the literature, from the database, from experiments, or from bioinformatics means. 
     In some embodiments, perturbation of the candidate genes in desired embryonic tissues containing a desired group of progenitor cells is performed using methods as described in above sections. 
     In some embodiments, the effect of candidate genes on the physiological and/or pathological status of the animal are evaluated. The perturbed candidate genes that produce a desired pathological condition or conditions therefore can be used as therapeutic targets. 
     In some embodiments, the effects of candidate genes on the progeny cells are measured based on the genomic, proteomic, genetic, epigenetic and/or phenotypic changes. In some embodiments, the effect of candidate genes on the progeny cells are measured using scRNA-Seq, and the changes in transcriptomic profile are evaluated against the candidate genes. The genes produce significant changes in gene expression programs that are pathologically relevant to the onset or status of the disease of interest therefore can be used as therapeutic targets. 
     In some embodiments, an experiment can be set up to test agents or compounds for their ability to modify the expression of the selected therapeutic target genes. In some embodiments, the agents of compounds can be antibodies, small molecules, peptides, or proteins. 
     Described in certain example embodiments herein are methods of in vivo screening for therapeutic targets useful for developing treatment for a disease, comprising:
         a. performing a of in vivo gene function analysis as further described elsewhere herein, wherein the plurality of genes are a plurality of candidate genes; and   b. selecting one or more candidate genes that produce a change in one or more identified gene modules that are indicative of the disease status; whereby the selected one or more candidate genes are identified as therapeutic targets for disease treatment screening.       

     In certain example embodiments, the method further comprises using the selected candidate gene(s) as therapeutic targets in a disease treatment screen. The term “candidate gene” refers to any gene that is being examined for the ability to modulate one or more phenotypic aspects of a cell or cell population as disclosed herein in a method performing an in vivo gene function analysis of the present invention and observing whether a modulation associated with particular cell state and/or disease takes place 
     In certain example embodiments, the disease treatment screen is an autism spectrum disease treatment screen. 
     In certain example embodiments, the disease is an autism spectrum disease. 
     Described in certain example embodiments herein are therapeutic agents for treating a disease where the therapeutic agent is capable of modifying the function, activity, expression, or a combination thereof of identified therapeutic targets identified using a method described elsewhere herein, one or more gene product(s) thereof, or both. 
     In certain exemplary embodiments, the disease is an autism spectrum disease. 
     Exemplary Therapies 
     The present invention also contemplates the uses of the in vivo genetic perturbation, in particular the in utero genetic perturbation described herein, for treatment in a variety of diseases and disorders. 
     In some embodiments, the invention described herein relates to a method for therapy in which a genetic abnormality or genetic abnormalities of an embryo can be corrected by in utero genetic perturbation described herein. The correction of genetic abnormality or abnormalities can be performed on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10, more than 20, or more than 50 genes in parallel as described herein. In some embodiments, the correction can be performed on multiple abnormalities on a single gene. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 abnormalities on a single gene can be corrected using the methods described herein. 
     In embodiments, the treatment is for disease/disorder of an organ, including brain diseases, liver disease, eye disease, muscle disease, heart disease, blood disease, kidney disease, or may comprise treatment for an autoimmune disease, central nervous system disease, cancer and other proliferative diseases, neurodegenerative disorders, inflammatory disease, metabolic disorder, musculoskeletal disorder and the like. 
     EXAMPLES 
     The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. 
     Example 1—In Vivo Pertub-Seq to Assess the Function of ASD Risk-Associated Genes 
     ASD/ND candidate genes from a recently published WES study of 11,986 cases with 6,430 ASD/ND probands were chosen (8) (Table 2). 38 candidate genes were initially prioritized (of which 35 were retained in the final analysis, Table 2) that harbor de novo variants specific to ASD/ND patients within the broader class of neurodevelopmental disability ( FIG. 5A , Table 2). These ASD/ND risk genes are expressed in human brain tissue, as assessed by the BrainSpan bulk RNA-seq dataset (9); some are highly expressed at embryonic stages, and others highly expressed from early postnatal to adult stages ( FIGS. 5B-5D ). Based on mouse cortical single-cell RNA sequencing (scRNA-seq) data, the orthologs of these ASD/ND risk genes are expressed in diverse cell types ( FIGS. 6A-6C ) (E18.5 data from the 10× Genomics public dataset (10); P7 data from this work). Thus, these ASD/ND genes could, in principle, act in many different cell types and temporal frames, requiring scalable methods to test gene function across a range of cell types and developmental events. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 ASD/ND risk gene list and their effect in the patient cohort. 
               
            
           
           
               
               
               
               
               
            
               
                 gene 
                 asd_rate_dn 
                 ddid_rate_dn 
                 qval_dnccPTV 
                 Alt_name_or_note 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 ADNP 
                 0.001399689 
                 0.003799392 
                 8.52E−15 
                   
               
               
                 ANK2 
                 0.001088647 
                 0.000379939 
                 1.43E−05 
               
               
                 ANKRD11 
                 0.000622084 
                 0.006079027 
                 9.55E−06 
                 Dropout in screen 
               
               
                 ARID1B 
                 0.001399689 
                 0.005889058 
                 2.58E−10 
               
               
                 ASH1L 
                 0.000933126 
                 0.000379939 
                 2.04E−05 
               
               
                 ASXL3 
                 0.000311042 
                 0.003419453 
                 0.019950532 
               
               
                 CHD2 
                 0.001088647 
                 0.001899696 
                 5.47E−06 
               
               
                 CHD8 
                 0.002954899 
                 0.000949848 
                 0 
               
               
                 CTNNB1 
                 0.000622084 
                 0.002849544 
                 3.98E−05 
               
               
                 CUL3 
                 0.000311042 
                 0.000759878 
                 0.301166206 
               
               
                 DDX3X 
                 0.000155521 
                 0.006079027 
                 0 
               
               
                 DSCAM 
                 0.000622084 
                 0.00018997 
                 0.000134664 
               
               
                 DYRK1A 
                 0.000933126 
                 0.003609422 
                 8.22E−10 
               
               
                 FBXO11 
                 0.000311042 
                 0.000949848 
                 0.530919852 
               
               
                 FOXP1 
                 0.001399689 
                 0.002659574 
                 1.77E−12 
               
               
                 GATAD2B 
                 0 
                 0.001899696 
                 0.923047848 
               
               
                 KDM5B 
                 0.000933126 
                 0.000569909 
                 0.000345432 
               
               
                 LARP4B 
                 0.000155521 
                 0.000379939 
                 0.514242847 
               
               
                 MAP1A 
                 0 
                 0.00018997 
                 0.009309407 
                 Dropout in screen 
               
               
                 MBD5 
                 0.000311042 
                 0.000569909 
                 0.008457933 
               
               
                 MED13L 
                 0.000777605 
                 0.003229483 
                 1.84E−06 
               
               
                 MLL1 
                 0.000466563 
                 0.005889058 
                 0.754464152 
                 KMT2A 
               
               
                 MYST4 
                 0 
                 0.002469605 
                 0.698791831 
                 KAT6B 
               
               
                 POGZ 
                 0.001088647 
                 0.002469605 
                 1.09E−10 
               
               
                 PTEN 
                 0.001088647 
                 0.000379939 
                 5.26E−08 
               
               
                 QRICH1 
                 0.000155521 
                 0.000569909 
                 0.103038387 
               
               
                 SATB2 
                 0.000311042 
                 0.002849544 
                 0.323451606 
               
               
                 SCN2A 
                 0.003421462 
                 0.00493921 
                 0 
               
               
                 SETD2 
                 0.000155521 
                 0.000569909 
                 0.704208112 
               
               
                 SETD5 
                 0.000466563 
                 0.003229483 
                 0.000184044 
               
               
                 SPEN 
                 0.000311042 
                 0.000569909 
                 0.758446035 
               
               
                 SUV420H1 
                 0.001088647 
                 0.000569909 
                 5.48E−10 
                 Dropout in screen 
               
               
                 SYNGAP1 
                 0.002177294 
                 0.003229483 
                 0 
               
               
                 TCF20 
                 0.000311042 
                 0.001709726 
                 0.029926217 
               
               
                 TCF7L2 
                 0.000311042 
                 0.000759878 
                 0.033914706 
               
               
                 TNRC6B 
                 0.000311042 
                 0.00018997 
                 0.3063045 
               
               
                 UPF3B 
                 0.000155521 
                 0.000569909 
                 0 
               
               
                 WAC 
                 0.000466563 
                 0.001139818 
                 0.000408636 
               
               
                   
               
            
           
         
       
     
     For in vivo Perturb-Seq, Cas9-mediated genome editing was used (11-13) in a pooled approach to introduce mutations in each of the ASD/ND risk genes within progenitor cells of the mouse developing forebrain in utero, followed by scRNA-seq at P7 to read out both a barcode identifying the perturbation and the expression profile of the perturbed cells ( FIG. 1A ). Specifically, a transgenic mouse line that constitutively expresses Cas9 (14) was used and pools of gRNAs targeting the different risk genes were delivered by lentiviral infection into the lateral ventricles of the developing embryo in utero. Each lentiviral vector contained two different gRNAs targeting the 5′-end coding exons of one ASD/ND gene (to enhance knockout efficiency), and a blue fluorescent protein (BFP) reporter with a unique barcode corresponding to the perturbation identity (11-13). To minimize vector recombination, packaged each lentivirus was packaged separately and then pooled viruses at equal titers. 
     A pool of lentiviruses with equal gRNA representation was injected into the ventricles of the developing forebrain at E12.5 ( FIG. 1A ). In this approach, lentiviral injection leads to infection of neural progenitors lining the lateral ventricle of the developing forebrain, including progenitors of the neocortex and the ganglionic eminences. Since lentiviral vectors integrate into the genome, the progeny of the infected progenitors are labeled by BFP and carry a perturbation barcode corresponding to the target ASD/ND gene. 
     Both immunohistochemical analysis and scRNA-seq of BFP+ cells at P7 showed that the Perturb-Seq vectors were expressed across a variety of neuronal and glial cell types in the cortex ( FIGS. 1B-1C and 7A-7B ). While microglia originate mostly from outside the targeted germinal zones, lentiviral vector expression was detected in cortical microglia, indicated by the presence of BFP as well as perturbation barcode expression, across multiple individual experiments ( FIGS. 8E-8F ). Without being bound by theory, it is possible that the in utero injection could have led to either local lesions that recruited and expanded the number of microglia along the injection tract, or that microglia were labeled within the parenchyma along the same tract. Overall, this approach allowed examination of the effects of each perturbation across a wide range of cell types from distinct brain regions (i.e., cortical projection neurons, interneurons, astroglia, oligodendroglia, etc.), and, importantly, under sparse labeling conditions where less than 0.1% of cells in the cortex were perturbed, and thus development of individual perturbed cells is highly unlikely to be affected by perturbed neighbors ( FIGS. 7A-7C ). 
     Example 2—In Vivo Perturb-Seq Targets Diverse Cell Types without Affecting Overall Cell Type Composition 
     The experiment was performed with 18 different cohorts of pregnant mice, for a total of 163 embryos, each subjected to the entire pool of perturbations. The cortical tissues were micro-dissected and dissociated separately at P7, FACS-enriched the perturbed cells by selecting for BFP expression and droplet-based scRNA-seq was used to obtain each cell&#39;s expression profile along with its perturbation barcode. The cell survival rate after FACS was 78%, and a 40-70% frameshift insertion/deletion for each gRNA target among the infected cells was confirmed ( FIGS. 7D-7E ). 
     This multiplexed experimental design allowed testing of the cell-autonomous effect of all perturbations against the effect of a negative control construct targeting the endogenous GFP in the Rosa26 locus, thus controlling for effects related to viral infection, among other confounders. To minimize batch-dependent variation, the control construct was included in the same pool as the perturbation vectors ( FIG. 7F ). After quality control, a total of 46,770 neocortical cells across 17 high-quality experimental batches was retained for further analysis. Cells were partitioned into major cell classes using Louvain clustering (15) and were annotated by known marker gene expression (16, 54) ( FIG. 1D ). 
     Five broad cell populations from this cortical dataset were focused on for downstream analysis: cortical projection neurons (8,450 cells), cortical inhibitory neurons (5,532 cells), astrocytes (9,526 cells), oligodendrocytes (4,279 cells), and microglia/macrophages (8,070 cells) (thus excluding vascular, endothelial, and contaminant hippocampal and striatal cells). Some remaining low-quality cells in these five major cell classes were further filtered out, retaining 35,857 high-quality cells (median of 2,436 detected genes per cell overall, and median of 4,084 genes in the projection neuron cluster, as expected from their large size and known high RNA content ( FIG. 7G )). Each of the five major cell types were subclustered separately and annotated biologically meaningful subclusters ( FIGS. 1E and 10A-10F ). 
     From inspecting the perturbation barcodes from the lentiviral constructs, 92% (33,231 cells) of the cells in these five major cell classes had at least one perturbation read assigned to them, and 50% had barcodes for a single gene ( FIGS. 8A-8C , 18,044 cells), reflecting the low multiplicity of infection ( FIG. 8D ). As it is rare for multiple ASD/ND loss-of-function risk gene mutations to co-occur in patients, the 18,044 cells that carried a single perturbation were focused on. A median of 338 cells per perturbation was found: after excluding perturbations with &lt;70 perturbed cells, 35 ASD/ND risk gene perturbations were retained. BFP from the lentiviral vector was robustly detected as one of the most highly expressed genes in all retained cells ( FIG. 8E ). The BFP detection rate in each cell type correlated with the average number of genes detected ( FIG. 8F ), further supporting the reliability of the readout. 
     ASD/ND risk gene perturbations had a very modest effect on the presence and proportions of these five major cell types relative to the negative control (targeting the GFP gene). Only loss of Dyrk1a had a significant effect on cell type composition, increasing the proportion of oligodendrocytes and reducing the proportion of microglia/macrophages [FDR-corrected P&lt;0.05 using Poisson regression (17)] ( FIGS. 1D and 9A-9C ). 
     Example 3—Co-Varying Gene Modules Associate with Cell States 
     To assess whether ASD/ND genetic perturbations caused molecular changes and alterations in cell states, it was first sought to define gene modules that co-vary within each of the five broad cell classes. As previous work has shown (11-13, 18), focusing on gene modules instead of individual genes provides more statistical power to detect biologically-meaningful perturbation effects using fewer cells than would be required for single gene-level analysis, and can capture diversity both within and across cell types. 
     It was first tested if the expression of known Gene Ontology (GO) gene sets (19) was affected by calculating a gene-set expression score for each cell and fitting a linear regression model to this score. After correcting for multiple hypothesis testing, no GO terms were significantly altered by any perturbation (Table 3). However, this approach is limited by the large number of tests performed (one test per GO term per cell type per perturbation, for a total of 510,265 tests), as well as the limited number of GO terms relevant to the developing cortex. 
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Analysis of ASD/ND risk gene perturbation effect for GO term gene signatures. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Estimate 
                 Std..Error 
                 t.value 
                 pval 
                 Pert 
                 Name 
                 CellType 
               
               
                   
               
               
                 −1.150003746 
                 0.226873197 
                 −5.068927323 
                 4.25E−07 
                 Pogz 
                 GO:0008528 
                 Astroglia 
               
               
                 −0.503415553 
                 0.101940391 
                 −4.938332582 
                 8.32E−07 
                 Cul3 
                 GO:0002026 
                 Astroglia 
               
               
                 −0.904979089 
                 0.19384124 
                 −4.668661265 
                 3.17E−06 
                 Chd8 
                 GO:0002162 
                 Astroglia 
               
               
                 −0.769374175 
                 0.165937695 
                 −4.63652442 
                  3.7E−06 
                 Upf3b 
                 GO:0008528 
                 Astroglia 
               
               
                 −0.737836517 
                 0.164941317 
                 −4.473327417 
                 7.99E−06 
                 Mbd5 
                 GO:0008528 
                 Astroglia 
               
               
                 −0.710936059 
                 0.159069379 
                 −4.469345775 
                 8.14E−06 
                 Tnrc6b 
                 GO:0008528 
                 Astroglia 
               
               
                 −0.389500932 
                 0.088034757 
                 −4.424399443 
                   1E−05 
                 Dyrk1a 
                 GO:0035082 
                 Astroglia 
               
               
                 0.913664946 
                 0.206578584 
                 4.422844472 
                 1.01E−05 
                 Larp4b 
                 GO:0010575 
                 Inhibitory 
               
               
                 −0.703224665 
                 0.159830257 
                 −4.39982191 
                 1.12E−05 
                 Stard9 
                 GO:0008528 
                 Astroglia 
               
               
                 −0.78708216 
                 0.179162272 
                 −4.393124476 
                 1.16E−05 
                 Dscam 
                 GO:0008528 
                 Astroglia 
               
               
                 0.713169269 
                 0.162340549 
                 4.393044591 
                 1.16E−05 
                 Tcf20 
                 GO:0043267 
                 Inhibitory 
               
               
                 −0.858063812 
                 0.196754612 
                 −4.361086136 
                 1.37E−05 
                 Myst4 
                 GO:0010524 
                 ODC 
               
               
                 0.418061159 
                 0.096423708 
                 4.335667709 
                  1.5E−05 
                 Arid1b 
                 GO:0017048 
                 Astroglia 
               
               
                 0.703614964 
                 0.16320145 
                 4.311327888 
                 1.68E−05 
                 Setd2 
                 GO:0008066 
                 Astroglia 
               
               
                 −0.403639559 
                 0.094133654 
                 −4.287941039 
                 1.86E−05 
                 Tnrc6b 
                 GO:0017134 
                 Microglia 
               
               
                 −0.456309221 
                 0.106709917 
                 −4.27616506 
                 1.96E−05 
                 Mll1 
                 GO:0002063 
                 Astroglia 
               
               
                 0.889502918 
                 0.208065792 
                 4.275104083 
                 1.97E−05 
                 Arid1b 
                 GO:0031681 
                 Microglia 
               
               
                 0.63742574 
                 0.150278773 
                 4.241621948 
                 2.29E−05 
                 Qrich1 
                 GO:0008066 
                 Astroglia 
               
               
                 −0.218032138 
                 0.051449362 
                 −4.237800626 
                 2.32E−05 
                 Kdm5b 
                 GO:0060021 
                 Excitatory 
               
               
                 −0.49174702 
                 0.11610971 
                 −4.235192907 
                 2.35E−05 
                 Kdm5b 
                 GO:0071498 
                 Astroglia 
               
               
                 −0.396324073 
                 0.093986743 
                 −4.216808261 
                 2.56E−05 
                 Ctnnb1 
                 GO:0017134 
                 Microglia 
               
               
                 0.384372831 
                 0.091194469 
                 4.214869977 
                 2.58E−05 
                 Arid1b 
                 GO:0030879 
                 Inhibitory 
               
               
                 −0.625053759 
                 0.148372438 
                 −4.212734986 
                  2.6E−05 
                 Stard9 
                 GO:0071385 
                 Astroglia 
               
               
                 0.711724638 
                 0.169879385 
                 4.18958802 
                 2.89E−05 
                 Chd2 
                 GO:0070886 
                 Inhibitory 
               
               
                 0.379939422 
                 0.09076425 
                 4.186002969 
                 2.93E−05 
                 Tcf20 
                 GO:0048468 
                 Inhibitory 
               
               
                 −0.72411368 
                 0.173909667 
                 −4.163734501 
                 3.22E−05 
                 Scn2a1 
                 GO:0008528 
                 Astroglia 
               
               
                 −0.587684917 
                 0.141005483 
                 −4.167816059 
                 3.23E−05 
                 Cul3 
                 GO:0016032 
                 ODC 
               
               
                 −0.601382071 
                 0.145300561 
                 −4.138883344 
                 3.59E−05 
                 Pogz 
                 GO:0004181 
                 Astroglia 
               
               
                 0.844335978 
                 0.204051793 
                 4.137851298 
                 3.61E−05 
                 Chd2 
                 GO:0008209 
                 Inhibitory 
               
               
                 −0.448797514 
                 0.108955995 
                 −4.119071322 
                 3.91E−05 
                 Upf3b 
                 GO:0071498 
                 Astroglia 
               
               
                 −0.74032323 
                 0.179596902 
                 −4.122138092 
                 3.93E−05 
                 Gatad2b 
                 GO:0050839 
                 ODC 
               
               
                 −0.45384813 
                 0.110388521 
                 −4.111370692 
                 4.04E−05 
                 Setd5 
                 GO:0051279 
                 Excitatory 
               
               
                 0.561042332 
                 0.136548137 
                 4.108751279 
                  4.1E−05 
                 Satb2 
                 GO:0042551 
                 Inhibitory 
               
               
                 0.353490322 
                 0.086213991 
                 4.100150295 
                 4.25E−05 
                 Adnp 
                 GO:0051018 
                 Inhibitory 
               
               
                 −0.252153048 
                 0.061682102 
                 −4.087945102 
                 4.46E−05 
                 Dyrk1a 
                 GO:0008047 
                 Excitatory 
               
               
                 −0.371646 
                 0.091206712 
                 −4.0747659 
                 4.73E−05 
                 Mll1 
                 GO:1904754 
                 Astroglia 
               
               
                 −0.18937733 
                 0.046497596 
                 −4.072841321 
                 4.76E−05 
                 Qrich1 
                 GO:0001889 
                 Excitatory 
               
               
                 −0.720056743 
                 0.176832653 
                 −4.07196709 
                 4.78E−05 
                 Kdm5b 
                 GO:0008528 
                 Astroglia 
               
               
                 −0.306595637 
                 0.075296366 
                 −4.071851707 
                  4.8E−05 
                 Mbd5 
                 GO:0006672 
                 Inhibitory 
               
               
                 −0.393088161 
                 0.096674939 
                 −4.066081309 
                 4.91E−05 
                 Mbd5 
                 GO:0002026 
                 Astroglia 
               
               
                 −0.501863493 
                 0.123590147 
                 −4.060707966 
                 5.03E−05 
                 Scn2a1 
                 GO:0097242 
                 Microglia 
               
               
                 −0.653181741 
                 0.161028176 
                 −4.056319564 
                 5.11E−05 
                 Asxl3 
                 GO:0008528 
                 Astroglia 
               
               
                 −0.375025774 
                 0.092542372 
                 −4.052476348 
                 5.21E−05 
                 Stard9 
                 GO:0030574 
                 Inhibitory 
               
               
                 0.45351895 
                 0.112147648 
                 4.043945256 
                 5.49E−05 
                 Dyrk1a 
                 GO:0006497 
                 ODC 
               
               
                 0.816094633 
                 0.202349586 
                 4.033092675 
                 5.64E−05 
                 Chd8 
                 GO:0043117 
                 Astroglia 
               
               
                 −0.388595264 
                 0.096415712 
                 −4.030414307 
                 5.71E−05 
                 Qrich1 
                 GO:0002026 
                 Astroglia 
               
               
                 −0.657359474 
                 0.163293172 
                 −4.025639681 
                 5.82E−05 
                 Wac 
                 GO:0008528 
                 Astroglia 
               
               
                 −0.374604107 
                 0.093225279 
                 −4.018267468 
                   6E−05 
                 Setd5 
                 GO:0007340 
                 Excitatory 
               
               
                 −1.161386124 
                 0.288841924 
                 −4.020836407 
                 6.05E−05 
                 Chd2 
                 GO:0032331 
                 ODC 
               
               
                 −0.458089532 
                 0.114200479 
                 −4.011275046 
                 6.19E−05 
                 Cul3 
                 GO:0071498 
                 Astroglia 
               
               
                 0.408514717 
                 0.101910067 
                 4.008580585 
                 6.28E−05 
                 Upf3b 
                 GO:2001171 
                 Inhibitory 
               
               
                 0.448587134 
                 0.111993288 
                 4.005482316 
                 6.36E−05 
                 Ctnnb1 
                 GO:0042551 
                 Inhibitory 
               
               
                 −0.343810608 
                 0.08597691 
                 −3.998871406 
                 6.52E−05 
                 Qrich1 
                 GO:0050807 
                 Astroglia 
               
               
                 −0.435745835 
                 0.109004224 
                 −3.997513302 
                 6.56E−05 
                 Setd2 
                 GO:0017134 
                 Microglia 
               
               
                 −0.462944572 
                 0.115859149 
                 −3.995753273 
                 6.72E−05 
                 Dyrk1a 
                 GO:0006672 
                 ODC 
               
               
                 −0.45795955 
                 0.114990881 
                 −3.982572752 
                 6.99E−05 
                 Ddx3x 
                 GO:0097242 
                 Microglia 
               
               
                 −0.2180048 
                 0.054831282 
                 −3.975920192 
                 7.17E−05 
                 Scn2a1 
                 GO:0035176 
                 Excitatory 
               
               
                 −0.570264328 
                 0.143566213 
                 −3.972134638 
                  7.3E−05 
                 Asxl3 
                 GO:0031994 
                 Microglia 
               
               
                 −0.385986815 
                 0.097258933 
                 −3.968651562 
                  7.4E−05 
                 Upf3b 
                 GO:0002026 
                 Astroglia 
               
               
                 0.396038929 
                 0.099806908 
                 3.96805127 
                 7.44E−05 
                 Myst4 
                 GO:0048468 
                 Inhibitory 
               
               
                 −0.557465429 
                 0.140629082 
                 −3.964083551 
                 7.55E−05 
                 Chd8 
                 GO:0097242 
                 Microglia 
               
               
                 0.430904319 
                 0.108896342 
                 3.95701371 
                 7.76E−05 
                 Kdm5b 
                 GO:0048535 
                 Excitatory 
               
               
                 0.495000995 
                 0.125188699 
                 3.954038961 
                 7.88E−05 
                 Ash1l 
                 GO:0042551 
                 Inhibitory 
               
               
                 −0.751987856 
                 0.190390415 
                 −3.949714888 
                 8.14E−05 
                 Ddx3x 
                 GO:0010524 
                 ODC 
               
               
                 0.331910936 
                 0.084179193 
                 3.942909445 
                 8.26E−05 
                 Mbd5 
                 GO:0072593 
                 Inhibitory 
               
               
                 0.270262663 
                 0.068750953 
                 3.931038802 
                 8.65E−05 
                 Med13l 
                 GO:0003746 
                 Excitatory 
               
               
                 −0.234093922 
                 0.059555666 
                 −3.930674216 
                 8.67E−05 
                 Ash1l 
                 GO:0030544 
                 Astroglia 
               
               
                 −0.299704412 
                 0.076342005 
                 −3.925812694 
                 8.84E−05 
                 Cul3 
                 GO:0010595 
                 Astroglia 
               
               
                 −0.491394061 
                 0.125404209 
                 −3.918481419 
                 9.12E−05 
                 Setd2 
                 GO:0007131 
                 Microglia 
               
               
                 −0.669874783 
                 0.170990817 
                 −3.917606783 
                 9.15E−05 
                 Ash1l 
                 GO:0008528 
                 Astroglia 
               
               
                 0.396104422 
                 0.101071583 
                 3.919048381 
                 9.23E−05 
                 Dyrk1a 
                 GO:0034497 
                 ODC 
               
               
                 0.356736465 
                 0.091158447 
                 3.913367086 
                 9.33E−05 
                 Scn2a1 
                 GO:0048468 
                 Inhibitory 
               
               
                 −0.484062687 
                 0.123771514 
                 −3.910937753 
                 9.41E−05 
                 Spen 
                 GO:0042129 
                 Microglia 
               
               
                 −0.662279697 
                 0.169396602 
                 −3.909639808 
                  9.6E−05 
                 Qrich1 
                 GO:0006851 
                 ODC 
               
               
                 −0.642420487 
                 0.164499039 
                 −3.905314522 
                 9.62E−05 
                 Qrich1 
                 GO:0008528 
                 Astroglia 
               
               
                 −0.34428546 
                 0.088275881 
                 −3.900107891 
                 9.83E−05 
                 Mll1 
                 GO:0046716 
                 Astroglia 
               
               
                 −0.979339536 
                 0.250883752 
                 −3.903559037 
                 9.84E−05 
                 Mbd5 
                 GO:2000279 
                 ODC 
               
               
                 −0.725737149 
                 0.186025221 
                 −3.901283627 
                 9.93E−05 
                 Tcf20 
                 GO:0010524 
                 ODC 
               
               
                 −0.335068585 
                 0.086068728 
                 −3.893035151 
                 0.000101186 
                 Asxl3 
                 GO:0004180 
                 Astroglia 
               
               
                 0.589982601 
                 0.151593062 
                 3.89188391 
                 0.000101665 
                 Upf3b 
                 GO:0008066 
                 Astroglia 
               
               
                 0.260763399 
                 0.067011696 
                 3.891311747 
                 0.000101904 
                 Ash1l 
                 GO:0000462 
                 Astroglia 
               
               
                 −0.28301018 
                 0.072753561 
                 −3.889983903 
                 0.00010246  
                 Mll1 
                 GO:0099645 
                 Astroglia 
               
               
                 −0.547951825 
                 0.140991211 
                 −3.886425413 
                 0.000104067 
                 Ctnnb1 
                 GO:0031994 
                 Microglia 
               
               
                 0.314413201 
                 0.080900692 
                 3.886409291 
                 0.000104213 
                 Med13l 
                 GO:0045739 
                 Inhibitory 
               
               
                 0.701726011 
                 0.180653534 
                 3.884374673 
                 0.000105085 
                 Dscam 
                 GO:0043267 
                 Inhibitory 
               
               
                 −0.294382572 
                 0.075800842 
                 −3.883631963 
                 0.000105161 
                 Ddx3x 
                 GO:0051019 
                 Astroglia 
               
               
                 −0.436520866 
                 0.112419901 
                 −3.88295011 
                 0.000105455 
                 Upf3b 
                 GO:0006959 
                 Astroglia 
               
               
                 −0.524930341 
                 0.13525899 
                 −3.880927542 
                 0.000106332 
                 Setd2 
                 GO:0060384 
                 Astroglia 
               
               
                 0.517783133 
                 0.13359741 
                 3.87569739 
                 0.00010863  
                 Dyrk1a 
                 GO:0043117 
                 Astroglia 
               
               
                 0.675503912 
                 0.174185958 
                 3.878061818 
                 0.000109202 
                 Mll1 
                 GO:0034383 
                 ODC 
               
               
                 −0.487435767 
                 0.125859896 
                 −3.87284418 
                 0.000109904 
                 Larp4b 
                 GO:0010595 
                 Astroglia 
               
               
                 −0.436328921 
                 0.112686892 
                 −3.872046822 
                 0.000110369 
                 Ddx3x 
                 GO:0032753 
                 Microglia 
               
               
                 −0.297908578 
                 0.076947207 
                 −3.871597022 
                 0.000110465 
                 Setd2 
                 GO:0043531 
                 Astroglia 
               
               
                 −0.943774819 
                 0.243567018 
                 −3.874805489 
                 0.00011066  
                 Qrich1 
                 GO:2000279 
                 ODC 
               
               
                 0.70157529 
                 0.181294661 
                 3.869806683 
                 0.000111529 
                 Pten 
                 GO:0005184 
                 Inhibitory 
               
               
                 −0.365114531 
                 0.094381379 
                 −3.868501754 
                 0.000111869 
                 Asxl3 
                 GO:0002026 
                 Astroglia 
               
               
                 −0.388885262 
                 0.100692524 
                 −3.862106598 
                 0.000115084 
                 Chd8 
                 GO:0050718 
                 Inhibitory 
               
               
                   
               
            
           
           
               
               
               
            
               
                 padj 
                 Ont 
                 Description 
               
               
                   
               
               
                 0.183522816 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.183522816 
                 Biological Process 
                 regulation of the force of heart contraction 
               
               
                 0.407867534 
                 Molecular Function 
                 dystroglycan binding 
               
               
                 0.407867534 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.465913758 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.465913758 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.465913758 
                 Biological Process 
                 axoneme assembly 
               
               
                 0.465913758 
                 Biological Process 
                 positive regulation of vascular endothelial growth factor 
               
               
                   
                   
                 production 
               
               
                 0.465913758 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.465913758 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.465913758 
                 Biological Process 
                 negative regulation of potassium ion transport 
               
               
                 0.498574515 
                 Biological Process 
                 positive regulation of calcium ion transport into cytosol 
               
               
                 0.498574515 
                 Molecular Function 
                 Rho GTPase binding 
               
               
                 0.498574515 
                 Molecular Function 
                 glutamate receptor activity 
               
               
                 0.498574515 
                 Molecular Function 
                 fibroblast growth factor binding 
               
               
                 0.498574515 
                 Biological Process 
                 chondrocyte development 
               
               
                 0.498574515 
                 Molecular Function 
                 G-protein beta-subunit binding 
               
               
                 0.498574515 
                 Molecular Function 
                 glutamate receptor activity 
               
               
                 0.498574515 
                 Biological Process 
                 roof of mouth development 
               
               
                 0.498574515 
                 Biological Process 
                 cellular response to fluid shear stress 
               
               
                 0.498574515 
                 Molecular Function 
                 fibroblast growth factor binding 
               
               
                 0.498574515 
                 Biological Process 
                 mammary gland development 
               
               
                 0.498574515 
                 Biological Process 
                 cellular response to glucocorticoid stimulus 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of calcineurin-NFAT signaling cascade 
               
               
                 0.514265412 
                 Biological Process 
                 cell development 
               
               
                 0.514265412 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.514265412 
                 Biological Process 
                 viral process 
               
               
                 0.514265412 
                 Molecular Function 
                 metallocarboxypeptidase activity 
               
               
                 0.514265412 
                 Biological Process 
                 androgen metabolic process 
               
               
                 0.514265412 
                 Biological Process 
                 cellular response to fluid shear stress 
               
               
                 0.514265412 
                 Molecular Function 
                 cell adhesion molecule binding 
               
               
                 0.514265412 
                 Biological Process 
                 regulation of release of sequestered calcium ion into cytosol 
               
               
                 0.514265412 
                 Biological Process 
                 neuron maturation 
               
               
                 0.514265412 
                 Molecular Function 
                 protein kinase A binding 
               
               
                 0.514265412 
                 Molecular Function 
                 enzyme activator activity 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of vascular associated smooth muscle 
               
               
                   
                   
                 cell migration 
               
               
                 0.514265412 
                 Biological Process 
                 liver development 
               
               
                 0.514265412 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.514265412 
                 Biological Process 
                 ceramide metabolic process 
               
               
                 0.514265412 
                 Biological Process 
                 regulation of the force of heart contraction 
               
               
                 0.514265412 
                 Biological Process 
                 amyloid-beta clearance 
               
               
                 0.514265412 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.514265412 
                 Biological Process 
                 collagen catabolic process 
               
               
                 0.514265412 
                 Biological Process 
                 protein lipidation 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of vascular permeability 
               
               
                 0.514265412 
                 Biological Process 
                 regulation of the force of heart contraction 
               
               
                 0.514265412 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.514265412 
                 Biological Process 
                 acrosome reaction 
               
               
                 0.514265412 
                 Biological Process 
                 negative regulation of chondrocyte differentiation 
               
               
                 0.514265412 
                 Biological Process 
                 cellular response to fluid shear stress 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of ATP biosynthetic process 
               
               
                 0.514265412 
                 Biological Process 
                 neuron maturation 
               
               
                 0.514265412 
                 Biological Process 
                 regulation of synapse organization 
               
               
                 0.514265412 
                 Molecular Function 
                 fibroblast growth factor binding 
               
               
                 0.514265412 
                 Biological Process 
                 ceramide metabolic process 
               
               
                 0.514265412 
                 Biological Process 
                 amyloid-beta clearance 
               
               
                 0.514265412 
                 Biological Process 
                 social behavior 
               
               
                 0.514265412 
                 Molecular Function 
                 insulin-like growth factor I binding 
               
               
                 0.514265412 
                 Biological Process 
                 regulation of the force of heart contraction 
               
               
                 0.514265412 
                 Biological Process 
                 cell development 
               
               
                 0.514265412 
                 Biological Process 
                 amyloid-beta clearance 
               
               
                 0.514265412 
                 Biological Process 
                 lymph node development 
               
               
                 0.514265412 
                 Biological Process 
                 neuron maturation 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of calcium ion transport into cytosol 
               
               
                 0.514265412 
                 Biological Process 
                 reactive oxygen species metabolic process 
               
               
                 0.514265412 
                 Molecular Function 
                 translation elongation factor activity 
               
               
                 0.514265412 
                 Molecular Function 
                 Hsp70 protein binding 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of endothelial cell migration 
               
               
                 0.514265412 
                 Biological Process 
                 reciprocal meiotic recombination 
               
               
                 0.514265412 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.514265412 
                 Biological Process 
                 protein localization to phagophore assembly site 
               
               
                 0.514265412 
                 Biological Process 
                 cell development 
               
               
                 0.514265412 
                 Biological Process 
                 regulation of T cell proliferation 
               
               
                 0.514265412 
                 Biological Process 
                 mitochondrial calcium ion transmembrane transport 
               
               
                 0.514265412 
                 Molecular Function 
                 G protein-coupled peptide receptor activity 
               
               
                 0.514265412 
                 Biological Process 
                 muscle cell cellular homeostasis 
               
               
                 0.514265412 
                 Biological Process 
                 negative regulation of DNA biosynthetic process 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of calcium ion transport into cytosol 
               
               
                 0.514265412 
                 Molecular Function 
                 carboxypeptidase activity 
               
               
                 0.514265412 
                 Molecular Function 
                 glutamate receptor activity 
               
               
                 0.514265412 
                 Biological Process 
                 maturation of SSU-rRNA from tricistronic rRNA transcript 
               
               
                   
                   
                 (SSU-rRNA, 5.8S rRNA, LSU-rRNA) 
               
               
                 0.514265412 
                 Biological Process 
                 neurotransmitter receptor localization to postsynaptic 
               
               
                   
                   
                 specialization membrane 
               
               
                 0.514265412 
                 Molecular Function 
                 insulin-like growth factor I binding 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of DNA repair 
               
               
                 0.514265412 
                 Biological Process 
                 negative regulation of potassium ion transport 
               
               
                 0.514265412 
                 Molecular Function 
                 mitogen-activated protein kinase binding 
               
               
                 0.514265412 
                 Biological Process 
                 humoral immune response 
               
               
                 0.514265412 
                 Biological Process 
                 innervation 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of vascular permeability 
               
               
                 0.514265412 
                 Biological Process 
                 low-density lipoprotein particle clearance 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of endothelial cell migration 
               
               
                 0.514265412 
                 Biological Process 
                 positive regulation of interleukin-4 production 
               
               
                 0.514265412 
                 Molecular Function 
                 ADP binding 
               
               
                 0.514265412 
                 Biological Process 
                 negative regulation of DNA biosynthetic process 
               
               
                 0.514265412 
                 Molecular Function 
                 neuropeptide hormone activity 
               
               
                 0.514265412 
                 Biological Process 
                 regulation of the force of heart contraction 
               
               
                 0.523591478 
                 Biological Process 
                 positive regulation of interleukin-1 beta secretion 
               
               
                   
               
            
           
         
       
     
     It was therefore sought to identify gene modules de novo in this data using two approaches: Weighted Gene Correlation Network Analysis (WGCNA), which identifies “modules” of genes with correlated expression, and structural topic modeling (STM), which attempts to reduce the dimensionality of the gene expression matrix and returns “topics” corresponding to the components of this representation ( FIGS. 2A, 10A-10F, 11A-11E, and 12A-12E  and Table 4) (20, 21). These analyses were performed for each of the five major cell clusters separately, to better identify effects associated with specific cell types; the nomenclature used here for the modules incorporates the cell cluster analysis it is derived from (e.g., PN1 represents a module identified by analysis of projection neurons). Each of these analyses used the full set of perturbations in order to identify effects shared across multiple perturbations. The subsequent analysis focused on the 14 modules identified by WGCNA, because they were highly correlated with one or more topics returned by STM ( FIGS. 11A-11E ). 
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 WGCNA gene module gene lists. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Module name in paper 
                 ODC1 
                 Mg1 
                 Mg2 
                 PN1 
                 PN2 
               
               
                 Note 
                 Progenitor 
                 Inflammatory 
                 Homeostatic 
                 Layer 4-5 
                 Neurite development 
               
               
                 Module name from WGCNA 
                 OPC_turquoise 
                 Mg_brown 
                 Mg_blue 
                 PN_greenyellow 
                 PN_brown 
               
               
                   
                 Pid1 
                 Marcksl1 
                 Serpine2 
                 Chgb 
                 Inpp4a 
               
               
                   
                 Ramp1 
                 Nfkbid 
                 Plxdc2 
                 Brinp1 
                 Pam 
               
               
                   
                 Dbi 
                 Egr2 
                 Cst3 
                 Hpca 
                 Dcaf6 
               
               
                   
                 Mdk 
                 Ccl2 
                 Gpr34 
                 Foxp1 
                 Nmt2 
               
               
                   
                 Stmn3 
                 Ccl7 
                 P2ry12 
                 Nrgn 
                 Yme1l1 
               
               
                   
                 Fabp7 
                 Ccl12 
                 Olfml3 
                 Nrsn1 
                 Set 
               
               
                   
                 Pea15a 
                 Ccl4 
                 Ldhb 
                 Fkbp1b 
                 Lrrc8a 
               
               
                   
                 Ddah1 
                 Nr4a1 
                 Pde3b 
                 Satb1 
                 Slc24a5 
               
               
                   
                 Car8 
                 Nfkbiz 
                 Cd81 
                 Rorb 
                 Myef2 
               
               
                   
                 Kcnd2 
                 Rcan1 
                 Sparc 
                   
                 Ncoa3 
               
               
                   
                 Apoe 
                 Tnf 
                 Hexb 
                   
                 Cask 
               
               
                   
                 Rlbp1 
                 Ier3 
                 Rhob 
                   
                 Skil 
               
               
                   
                 Mmp15 
                   
                 Lgmn 
                   
                 Rap2b 
               
               
                   
                 Cspg5 
                   
                   
                   
                 Slc39a1 
               
               
                   
                 Rgcc 
                   
                   
                   
                 Rbm15 
               
               
                   
                 6330403K07Rik 
                   
                   
                   
                 Klhl9 
               
               
                   
                 Sox11 
                   
                   
                   
                 Ythdf2 
               
               
                   
                 Tspan13 
                   
                   
                   
                 Gnb1 
               
               
                   
                 Zfp36l2 
                   
                   
                   
                 Gatad1 
               
               
                   
                 1700086L19Rik 
                   
                   
                   
                 Insig1 
               
               
                   
                 Hes1 
                   
                   
                   
                 Actb 
               
               
                   
                 Pcp4 
                   
                   
                   
                 Kdelr2 
               
               
                   
                 Ppp2r2b 
                   
                   
                   
                 Cbx3 
               
               
                   
                 Cdo1 
                   
                   
                   
                 Sfxn5 
               
               
                   
                 Cspg4 
                   
                   
                   
                 Iqsec1 
               
               
                   
                 Ostf1 
                   
                   
                   
                 Tmf1 
               
               
                   
                   
                   
                   
                   
                 Dpf1 
               
               
                   
                   
                   
                   
                   
                 Shank1 
               
               
                   
                   
                   
                   
                   
                 Klf13 
               
               
                   
                   
                   
                   
                   
                 Abhd2 
               
               
                   
                   
                   
                   
                   
                 Mcmbp 
               
               
                   
                   
                   
                   
                   
                 Sec23ip 
               
               
                   
                   
                   
                   
                   
                 Hsf2 
               
               
                   
                   
                   
                   
                   
                 Sirt6 
               
               
                   
                   
                   
                   
                   
                 Ppm1h 
               
               
                   
                   
                   
                   
                   
                 Micu3 
               
               
                   
                   
                   
                   
                   
                 Lzts1 
               
               
                   
                   
                   
                   
                   
                 Ncan 
               
               
                   
                   
                   
                   
                   
                 Upf1 
               
               
                   
                   
                   
                   
                   
                 Supt16 
               
               
                   
                   
                   
                   
                   
                 Dpysl2 
               
               
                   
                   
                   
                   
                   
                 Spry2 
               
               
                   
                   
                   
                   
                   
                 Zbtb44 
               
               
                   
                   
                   
                   
                   
                 Scn2b 
               
               
                   
                   
                   
                   
                   
                 Ncam1 
               
               
                   
                   
                   
                   
                   
                 AI593442 
               
               
                   
                   
                   
                   
                   
                 Dapk2 
               
               
                   
                   
                   
                   
                   
                 Clasp2 
               
               
                   
                   
                   
                   
                   
                 Actr2 
               
               
                   
                   
                   
                   
                   
                 Alkbh5 
               
               
                   
                   
                   
                   
                   
                 Arhgap44 
               
               
                   
                   
                   
                   
                   
                 Gas7 
               
               
                   
                   
                   
                   
                   
                 Kdm6b 
               
               
                   
                   
                   
                   
                   
                 Dusp3 
               
               
                   
                   
                   
                   
                   
                 Fn3krp 
               
               
                   
                   
                   
                   
                   
                 Zscan26 
               
               
                   
                   
                   
                   
                   
                 Atxn1 
               
               
                   
                   
                   
                   
                   
                 Hmgcr 
               
               
                   
                   
                   
                   
                   
                 Map1b 
               
               
                   
                   
                   
                   
                   
                 Paip1 
               
               
                   
                   
                   
                   
                   
                 Dbpht2 
               
               
                   
                   
                   
                   
                   
                 St13 
               
               
                   
                   
                   
                   
                   
                 Kmt2d 
               
               
                   
                   
                   
                   
                   
                 Mkl2 
               
               
                   
                   
                   
                   
                   
                 Rrn3 
               
               
                   
                   
                   
                   
                   
                 Marf1 
               
               
                   
                   
                   
                   
                   
                 Lrrc58 
               
               
                   
                   
                   
                   
                   
                 Gsk3b 
               
               
                   
                   
                   
                   
                   
                 Tmem181a 
               
               
                   
                   
                   
                   
                   
                 D17H6S53E 
               
               
                   
                   
                   
                   
                   
                 Birc6 
               
               
                   
                   
                   
                   
                   
                 Dctn4 
               
               
                   
                   
                   
                   
                   
                 Otub1 
               
               
                   
                   
                   
                   
                   
                 Ric1 
               
               
                   
                   
                   
                   
                   
                 Pdcd4 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Module name in paper 
                 PN3 
                 PN4 
                 PN5 
                 PN6 
                 IN1 
               
               
                 Note 
                 Layer 4-5 
                 Neurotransmitter/Layer 6 
                 Tubulin and ATP biogenesis 
                 Layer 5-6 
                 Ndnf+ 
               
               
                 Module name from WGCNA 
                 PN_purple 
                 PN_red 
                 PN_yellow 
                 PN_magenta 
                 IN_blue 
               
               
                   
                 Syndig1 
                 Etl4 
                 Arpc2 
                 Tmem163 
                 A830018L16Rik 
               
               
                   
                 Lhfp 
                 Olfm1 
                 Atp1b1 
                 Serpini1 
                 Kcnq5 
               
               
                   
                 Slc30a3 
                 Trp53i11 
                 Atp5c1 
                 Grik3 
                 Spats2l 
               
               
                   
                 Plb1 
                 Slc1a2 
                 Gnas 
                 Galnt9 
                 Lancl1 
               
               
                   
                 Plxnd1 
                 Rasgrp1 
                 Atp5f1 
                 Crym 
                 Erbb4 
               
               
                   
                 Cox6a2 
                 Syt11 
                 Dnaja1 
                 Hs3st4 
                 43894 
               
               
                   
                 Rora 
                 Sh3gl2 
                 Ywhah 
                 Fezf2 
                 Resp18 
               
               
                   
                 Krt12 
                 Elavl2 
                 Gnb2 
                 Nxph3 
                 Nyap2 
               
               
                   
                 A830036E02Rik 
                 Nfia 
                 Gapdh 
                 Nptx1 
                 Irs1 
               
               
                   
                 Dcdc2a 
                 Fxyd7 
                 Aldoa 
                 Bcl11b 
                 Lefty2 
               
               
                   
                 A830009L08Rik 
                 Slc17a7 
                 Atp5b 
                 Tle4 
                 Susd4 
               
               
                   
                   
                 Gsg1l 
                 Tecr 
                   
                 Tgfb2 
               
               
                   
                   
                 Ipcef1 
                 Tubb3 
                   
                 Fam163b 
               
               
                   
                   
                 Nxph4 
                 Hspa8 
                   
                 Rapgef4 
               
               
                   
                   
                 Cplx3 
                 Eif4a1 
                   
                 Nckap1 
               
               
                   
                   
                 Rab6b 
                 Ywhae 
                   
                 Lamp5 
               
               
                   
                   
                 Sez6 
                 Psmc5 
                   
                 Pak7 
               
               
                   
                   
                 Pcp4 
                 Actg1 
                   
                 Tox2 
               
               
                   
                   
                 Clic5 
                 Tubb2a 
                   
                 Dok5 
               
               
                   
                   
                 Pcsk5 
                 Tubb2b 
                   
                 Chrna4 
               
               
                   
                   
                   
                 Hsp90aa1 
                   
                 Atp6ap2 
               
               
                   
                   
                   
                 Tuba1b 
                   
                 Trpc5 
               
               
                   
                   
                   
                 Tuba1a 
                   
                 Frmpd4 
               
               
                   
                   
                   
                 Ap2m1 
                   
                 Rxfp1 
               
               
                   
                   
                   
                 Eif4a2 
                   
                 Man1a2 
               
               
                   
                   
                   
                 Tubb5 
                   
                 Unc5c 
               
               
                   
                   
                   
                 Hsp90ab1 
                   
                 Lingo2 
               
               
                   
                   
                   
                 Vapa 
                   
                 Gabbr2 
               
               
                   
                   
                   
                 Tmem178 
                   
                 Dab1 
               
               
                   
                   
                   
                   
                   
                 Cacna2d1 
               
               
                   
                   
                   
                   
                   
                 Reln 
               
               
                   
                   
                   
                   
                   
                 Afap1 
               
               
                   
                   
                   
                   
                   
                 Limch1 
               
               
                   
                   
                   
                   
                   
                 Kit 
               
               
                   
                   
                   
                   
                   
                 Hopx 
               
               
                   
                   
                   
                   
                   
                 Parm1 
               
               
                   
                   
                   
                   
                   
                 Rimbp2 
               
               
                   
                   
                   
                   
                   
                 Col26a1 
               
               
                   
                   
                   
                   
                   
                 Nxph1 
               
               
                   
                   
                   
                   
                   
                 Ndufa4 
               
               
                   
                   
                   
                   
                   
                 Npy 
               
               
                   
                   
                   
                   
                   
                 Osbpl3 
               
               
                   
                   
                   
                   
                   
                 Ndnf 
               
               
                   
                   
                   
                   
                   
                 RP23-291B1.2 
               
               
                   
                   
                   
                   
                   
                 Ldha 
               
               
                   
                   
                   
                   
                   
                 Adamts17 
               
               
                   
                   
                   
                   
                   
                 Homer2 
               
               
                   
                   
                   
                   
                   
                 Hs3st4 
               
               
                   
                   
                   
                   
                   
                 Rgs10 
               
               
                   
                   
                   
                   
                   
                 Ppp1r14c 
               
               
                   
                   
                   
                   
                   
                 Sgk1 
               
               
                   
                   
                   
                   
                   
                 Hs3st5 
               
               
                   
                   
                   
                   
                   
                 Nav3 
               
               
                   
                   
                   
                   
                   
                 Ppm1h 
               
               
                   
                   
                   
                   
                   
                 Zmat4 
               
               
                   
                   
                   
                   
                   
                 Mfap3l 
               
               
                   
                   
                   
                   
                   
                 Abhd8 
               
               
                   
                   
                   
                   
                   
                 Rasd2 
               
               
                   
                   
                   
                   
                   
                 Psmb10 
               
               
                   
                   
                   
                   
                   
                 Maf 
               
               
                   
                   
                   
                   
                   
                 Cpne7 
               
               
                   
                   
                   
                   
                   
                 Ube2e2 
               
               
                   
                   
                   
                   
                   
                 Gdf10 
               
               
                   
                   
                   
                   
                   
                 Fgf9 
               
               
                   
                   
                   
                   
                   
                 Pnoc 
               
               
                   
                   
                   
                   
                   
                 Slitrk1 
               
               
                   
                   
                   
                   
                   
                 B3gat1 
               
               
                   
                   
                   
                   
                   
                 Sorl1 
               
               
                   
                   
                   
                   
                   
                 Cryab 
               
               
                   
                   
                   
                   
                   
                 Cck 
               
               
                   
                   
                   
                   
                   
                 Asic2 
               
               
                   
                   
                   
                   
                   
                 Hecw1 
               
               
                   
                   
                   
                   
                   
                 Amph 
               
               
                   
                   
                   
                   
                   
                 A330102I10Rik 
               
               
                   
                   
                   
                   
                   
                 Mctp1 
               
               
                   
                   
                   
                   
                   
                 Hapln1 
               
               
                   
                   
                   
                   
                   
                 Sv2c 
               
               
                   
                   
                   
                   
                   
                 Id2 
               
               
                   
                   
                   
                   
                   
                 Sstr1 
               
               
                   
                   
                   
                   
                   
                 Rgs6 
               
               
                   
                   
                   
                   
                   
                 Sema5a 
               
               
                   
                   
                   
                   
                   
                 Csmd3 
               
               
                   
                   
                   
                   
                   
                 Nov 
               
               
                   
                   
                   
                   
                   
                 Mpped1 
               
               
                   
                   
                   
                   
                   
                 Grin2a 
               
               
                   
                   
                   
                   
                   
                 Il1rap 
               
               
                   
                   
                   
                   
                   
                 Ptprm 
               
               
                   
                   
                   
                   
                   
                 Fkbp2 
               
               
                   
                   
                   
                   
                   
                 Cox8a 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Module name in paper 
                 IN2 
                 Astro1 
                 Astro2 
                 Astro3 
               
               
                   
                 Note 
                 Vip+ 
                 Homeostatic1 
                 Activation 
                 Homeostatic2 
               
               
                   
                 Module name from WGCNA 
                 IN_green 
                 Astro_blue 
                 Astro_green 
                 Astro_turquoise 
               
               
                   
                   
                 Asic4 
                 Hsd11b1 
                 Bgn 
                 Prex2 
               
               
                   
                   
                 Pam 
                 Chst1 
                 S100a4 
                 Col9a1 
               
               
                   
                   
                 Prox1 
                 Kcnip3 
                 S100a11 
                 Arhgef4 
               
               
                   
                   
                 Ap1s2 
                 Phactr3 
                 S100a10 
                 Bmpr2 
               
               
                   
                   
                 Cnr1 
                 Tspan7 
                 Igfbp7 
                 Nrp2 
               
               
                   
                   
                 Klhl9 
                 Chrdl1 
                 Ifitm3 
                 Mgat5 
               
               
                   
                   
                 Sema3c 
                 Fam212b 
                 C1ql1 
                 Adora1 
               
               
                   
                   
                 Npas1 
                 St6galnac5 
                 Timp2 
                 Rgs2 
               
               
                   
                   
                 Nr2f2 
                 Cth 
                 S1pr3 
                 Glul 
               
               
                   
                   
                 Igf1 
                 Gabbr2 
                 Ntrk2 
                 Tnr 
               
               
                   
                   
                 Adra1b 
                 Efhd2 
                 Actn1 
                 Myoc 
               
               
                   
                   
                 Adarb2 
                 AW011738 
                 Serpina3n 
                 Ccdc3 
               
               
                   
                   
                 Cxcl14 
                 Grm3 
                 Ccdc74a 
                 Nsmf 
               
               
                   
                   
                   
                 0610040J01Rik 
                 C4b 
                 Ggta1 
               
               
                   
                   
                   
                 Abcb9 
                   
                 Nr4a2 
               
               
                   
                   
                   
                 Ptprz1 
                   
                 Pkp4 
               
               
                   
                   
                   
                 Akr1b10 
                   
                 Ssfa2 
               
               
                   
                   
                   
                 Timp4 
                   
                 Slc1a2 
               
               
                   
                   
                   
                 Eps8 
                   
                 Cd44 
               
               
                   
                   
                   
                 Slco1c1 
                   
                 Serf2 
               
               
                   
                   
                   
                 Aplp1 
                   
                 Slc20a1 
               
               
                   
                   
                   
                 Slc7a10 
                   
                 Btbd3 
               
               
                   
                   
                   
                 Grm5 
                   
                 Ptprt 
               
               
                   
                   
                   
                 Dhcr7 
                   
                 Matn4 
               
               
                   
                   
                   
                 Ccnd1 
                   
                 Elmo2 
               
               
                   
                   
                   
                 S100b 
                   
                 Sulf2 
               
               
                   
                   
                   
                 Gpc5 
                   
                 Usp9x 
               
               
                   
                   
                   
                 Cadm1 
                   
                 Slc6a8 
               
               
                   
                   
                   
                 Arpp21 
                   
                 Plp1 
               
               
                   
                   
                   
                 Rnf215 
                   
                 Gpm6b 
               
               
                   
                   
                   
                 Slc13a5 
                   
                 Trim2 
               
               
                   
                   
                   
                 Etv4 
                   
                 Slc16a1 
               
               
                   
                   
                   
                 Dbx2 
                   
                 Unc5c 
               
               
                   
                   
                   
                 Olig2 
                   
                 Bmpr1b 
               
               
                   
                   
                   
                 Cbr3 
                   
                 Grin3a 
               
               
                   
                   
                   
                 Hbegf 
                   
                 Rspo1 
               
               
                   
                   
                   
                 Cdo1 
                   
                 Man1c1 
               
               
                   
                   
                   
                 Vldlr 
                   
                 Ptchd2 
               
               
                   
                   
                   
                   
                   
                 Ski 
               
               
                   
                   
                   
                   
                   
                 Agrn 
               
               
                   
                   
                   
                   
                   
                 Prkag2 
               
               
                   
                   
                   
                   
                   
                 Hopx 
               
               
                   
                   
                   
                   
                   
                 Miat 
               
               
                   
                   
                   
                   
                   
                 Hspb8 
               
               
                   
                   
                   
                   
                   
                 Clip2 
               
               
                   
                   
                   
                   
                   
                 Tsc22d4 
               
               
                   
                   
                   
                   
                   
                 D630045J12Rik 
               
               
                   
                   
                   
                   
                   
                 Gadd45a 
               
               
                   
                   
                   
                   
                   
                 Atoh8 
               
               
                   
                   
                   
                   
                   
                 Tmem150a 
               
               
                   
                   
                   
                   
                   
                 Tgoln1 
               
               
                   
                   
                   
                   
                   
                 Ctnna2 
               
               
                   
                   
                   
                   
                   
                 Slc6a6 
               
               
                   
                   
                   
                   
                   
                 Adamts9 
               
               
                   
                   
                   
                   
                   
                 A2m 
               
               
                   
                   
                   
                   
                   
                 Kcna6 
               
               
                   
                   
                   
                   
                   
                 Zfp36 
               
               
                   
                   
                   
                   
                   
                 Clip3 
               
               
                   
                   
                   
                   
                   
                 Nav2 
               
               
                   
                   
                   
                   
                   
                 Arhgef17 
               
               
                   
                   
                   
                   
                   
                 Tab2 
               
               
                   
                   
                   
                   
                   
                 Marcks 
               
               
                   
                   
                   
                   
                   
                 Sgpl1 
               
               
                   
                   
                   
                   
                   
                 Slc7a2 
               
               
                   
                   
                   
                   
                   
                 Mfap3l 
               
               
                   
                   
                   
                   
                   
                 Ncan 
               
               
                   
                   
                   
                   
                   
                 Adgrl1 
               
               
                   
                   
                   
                   
                   
                 Zfp423 
               
               
                   
                   
                   
                   
                   
                 Mt3 
               
               
                   
                   
                   
                   
                   
                 Mt2 
               
               
                   
                   
                   
                   
                   
                 Mt1 
               
               
                   
                   
                   
                   
                   
                 Cadps 
               
               
                   
                   
                   
                   
                   
                 Opcml 
               
               
                   
                   
                   
                   
                   
                 Sorl1 
               
               
                   
                   
                   
                   
                   
                 Cryab 
               
               
                   
                   
                   
                   
                   
                 Islr 
               
               
                   
                   
                   
                   
                   
                 6030419C18Rik 
               
               
                   
                   
                   
                   
                   
                 Smad6 
               
               
                   
                   
                   
                   
                   
                 Igdcc4 
               
               
                   
                   
                   
                   
                   
                 Pth1r 
               
               
                   
                   
                   
                   
                   
                 Tmie 
               
               
                   
                   
                   
                   
                   
                 Csrnp1 
               
               
                   
                   
                   
                   
                   
                 Tmem158 
               
               
                   
                   
                   
                   
                   
                 Nsg2 
               
               
                   
                   
                   
                   
                   
                 Wwc1 
               
               
                   
                   
                   
                   
                   
                 Col23a1 
               
               
                   
                   
                   
                   
                   
                 Slc36a2 
               
               
                   
                   
                   
                   
                   
                 Serpinf1 
               
               
                   
                   
                   
                   
                   
                 Taok1 
               
               
                   
                   
                   
                   
                   
                 Nog 
               
               
                   
                   
                   
                   
                   
                 Stat3 
               
               
                   
                   
                   
                   
                   
                 Dusp3 
               
               
                   
                   
                   
                   
                   
                 Prkca 
               
               
                   
                   
                   
                   
                   
                 Fasn 
               
               
                   
                   
                   
                   
                   
                 Aldh5a1 
               
               
                   
                   
                   
                   
                   
                 Mylip 
               
               
                   
                   
                   
                   
                   
                 Msx2 
               
               
                   
                   
                   
                   
                   
                 Erbb2ip 
               
               
                   
                   
                   
                   
                   
                 Map3k1 
               
               
                   
                   
                   
                   
                   
                 Id2 
               
               
                   
                   
                   
                   
                   
                 Rgs6 
               
               
                   
                   
                   
                   
                   
                 Lifr 
               
               
                   
                   
                   
                   
                   
                 Ank 
               
               
                   
                   
                   
                   
                   
                 Enpp2 
               
               
                   
                   
                   
                   
                   
                 Plec 
               
               
                   
                   
                   
                   
                   
                 Maff 
               
               
                   
                   
                   
                   
                   
                 Shisa8 
               
               
                   
                   
                   
                   
                   
                 3-Sep 
               
               
                   
                   
                   
                   
                   
                 Fam19a5 
               
               
                   
                   
                   
                   
                   
                 Slc38a1 
               
               
                   
                   
                   
                   
                   
                 Snai2 
               
               
                   
                   
                   
                   
                   
                 Nfkbiz 
               
               
                   
                   
                   
                   
                   
                 Robo2 
               
               
                   
                   
                   
                   
                   
                 Adamts1 
               
               
                   
                   
                   
                   
                   
                 Tulp4 
               
               
                   
                   
                   
                   
                   
                 Paqr4 
               
               
                   
                   
                   
                   
                   
                 Fgd2 
               
               
                   
                   
                   
                   
                   
                 H2-DMa 
               
               
                   
                   
                   
                   
                   
                 H2-DMb1 
               
               
                   
                   
                   
                   
                   
                 H2-Ab1 
               
               
                   
                   
                   
                   
                   
                 H2-Aa 
               
               
                   
                   
                   
                   
                   
                 H2-Eb1 
               
               
                   
                   
                   
                   
                   
                 Ddr1 
               
               
                   
                   
                   
                   
                   
                 Npc1 
               
               
                   
                   
                   
                   
                   
                 Aqp4 
               
               
                   
                   
                   
                   
                   
                 Nrep 
               
               
                   
                   
                   
                   
                   
                 Cd74 
               
               
                   
                   
                   
                   
                   
                 Cidea 
               
               
                   
                   
                   
                   
                   
                 Smad7 
               
               
                   
                   
                   
                   
                   
                 Fth1 
               
               
                   
                   
                   
                   
                   
                 Aldh1a1 
               
               
                   
                   
                   
                   
                   
                 Kank1 
               
               
                   
                   
                   
                   
                   
                 Scd2 
               
               
                   
                   
               
            
           
         
       
     
     The 14 WGCNA modules comprised two broad categories. Some reflect common biological processes and were present across multiple cell subsets (e.g., cell cycle, differentiation, maturation). For example, module PN2 is associated with genes involved in neurite development and varied across cells in multiple projection neuron subclusters ( FIG. 10A ). Others represent cell type-specific features unique to only some subsets (e.g., subcluster-specific features of a neuronal sub-type). For example, module PN1 is a module associated with two defined subclusters of projection neurons of Layer 4 and Layer 5 ( FIG. 10A ). 
     Example 4—ASD/ND Gene Perturbations Affect Cell States in Multiple Cell Classes 
     As the WGCNA analysis is expected to recover gene modules associated with many kinds of variation across the data, the association of each risk gene perturbation with the 14 individual WGCNA gene modules was tested next. It was estimated the effect size of each perturbation on each gene module by fitting a joint linear regression model, estimating how module gene expression in cells from each perturbation group deviated from the GFP control cells ( FIG. 2A-2B ). To ensure that no single perturbation or batch dominated the linear model, the cells in each cell category were down-sampled such that no perturbation had more than two times the median number of cells over all perturbations. This linear regression analysis was performed on mean-centered and standard deviation-scaled module scores, so effect sizes can be interpreted in terms of standard deviations from the population mean ( FIG. 2B ). This modeling approach assumes that module expression in individual cells is independent after conditioning on the experimental batch, and that noise is normally distributed. To evaluate the effects of these assumptions, alternative approaches were also compared, including a linear mixed model-based approach and a permutation-based approach ( FIGS. 13A-13E  and Table 5). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Alternative effect size and statistical measurements of Perturb-Seq. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 perturbation 
                 Module 
                 pval 
                 padj 
                 Estimate 
                 SE 
                 df 
                 t.value 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Gatad2b 
                 ODC1 
                 0.00019929 
                 0.09148041 
                 −1.6392731 
                 0.43402622 
                 245.473281 
                 −3.7768988 
               
               
                 Ash1l 
                 PN1 
                 0.00040956 
                 0.09148041 
                 −0.5702511 
                 0.15798048 
                 160.261013 
                 −3.6096302 
               
               
                 Chd8 
                 ODC1 
                 0.00061256 
                 0.09148041 
                 −1.4433344 
                 0.4157381 
                 241.328595 
                 −3.4717395 
               
               
                 Stard9 
                 PN3 
                 0.00074678 
                 0.09148041 
                 −0.4945026 
                 0.14236812 
                 105.399114 
                 −3.4734084 
               
               
                 Kdm5b 
                 ODC1 
                 0.00105686 
                 0.0947702 
                 −1.2177755 
                 0.36673854 
                 213.093268 
                 −3.320555 
               
               
                 Adnp 
                 PN1 
                 0.00174366 
                 0.0947702 
                 −0.5197172 
                 0.16287405 
                 142.956625 
                 −3.1909144 
               
               
                 Ash1l 
                 PN3 
                 0.0017729 
                 0.0947702 
                 −0.4682456 
                 0.14676601 
                 132.809402 
                 −3.1904225 
               
               
                 Fbxo11 
                 PN3 
                 0.00183349 
                 0.0947702 
                 −0.4707365 
                 0.14841361 
                 151.862298 
                 −3.171788 
               
               
                 Setd2 
                 ODC1 
                 0.00192802 
                 0.0947702 
                 −1.1736475 
                 0.37333025 
                 195.532048 
                 −3.1437246 
               
               
                 Upf3b 
                 ODC1 
                 0.00193409 
                 0.0947702 
                 −1.1332804 
                 0.36093759 
                 209.827187 
                 −3.1398237 
               
               
                 Stard9 
                 PN1 
                 0.002176 
                 0.09693093 
                 −0.4832762 
                 0.15463063 
                 134.549616 
                 −3.1253587 
               
               
                 Asxl3 
                 PN3 
                 0.0024178 
                 0.09872663 
                 −0.4535802 
                 0.14641887 
                 122.498726 
                 −3.0978265 
               
               
                 Setd5 
                 ODC1 
                 0.00374726 
                 0.14124293 
                 −1.0649438 
                 0.3633284 
                 212.539974 
                 −2.9310778 
               
               
                 Cul3 
                 ODC1 
                 0.00470021 
                 0.14418933 
                 −1.03766 
                 0.36318713 
                 212.478167 
                 −2.8570945 
               
               
                 Adnp 
                 ODC1 
                 0.0047499 
                 0.14418933 
                 −1.0526633 
                 0.36915875 
                 228.302171 
                 −2.8515192 
               
               
                 Ctnnb1 
                 PN1 
                 0.00481414 
                 0.14418933 
                 −0.4243003 
                 0.14816893 
                 144.072277 
                 −2.8636256 
               
               
                 Scn2a1 
                 PN1 
                 0.0055001 
                 0.14418933 
                 −0.4711114 
                 0.16753867 
                 170.957327 
                 −2.811956 
               
               
                 Upf3b 
                 PN3 
                 0.00556665 
                 0.14418933 
                 −0.4351463 
                 0.15448305 
                 137.233728 
                 −2.8167896 
               
               
                 Setd5 
                 PN3 
                 0.00585783 
                 0.14418933 
                 −0.4603121 
                 0.16512846 
                 187.205554 
                 −2.7876001 
               
               
                 Scn2a1 
                 PN3 
                 0.00588528 
                 0.14418933 
                 −0.4364571 
                 0.15614299 
                 145.694403 
                 −2.7952399 
               
               
                 Kdm5b 
                 PN1 
                 0.00685531 
                 0.14996152 
                 −0.4831399 
                 0.17729142 
                 265.365193 
                 −2.725117 
               
               
                 Cul3 
                 PN1 
                 0.00716729 
                 0.14996152 
                 −0.4419764 
                 0.16134234 
                 111.695992 
                 −2.73937 
               
               
                 Ctnnb1 
                 ODC1 
                 0.00735312 
                 0.14996152 
                 −0.9658219 
                 0.35653152 
                 194.185047 
                 −2.7089383 
               
               
                 Ank2 
                 PN1 
                 0.00763061 
                 0.14996152 
                 −0.5417298 
                 0.20070335 
                 175.6413 
                 −2.699157 
               
               
                 Ash1l 
                 ODC1 
                 0.00825021 
                 0.14996152 
                 −0.9879851 
                 0.37022521 
                 197.370732 
                 −2.6686057 
               
               
                 Ank2 
                 IN1 
                 0.00838415 
                 0.14996152 
                 0.50499251 
                 0.19081393 
                 509.391877 
                 2.64651802 
               
               
                 Dscam 
                 IN1 
                 0.00849347 
                 0.14996152 
                 0.39991241 
                 0.15054164 
                 213.047921 
                 2.65649027 
               
               
                 Syngap1 
                 PN3 
                 0.00860101 
                 0.14996152 
                 −0.4422123 
                 0.16644951 
                 179.551063 
                 −2.6567356 
               
               
                 Mbd5 
                 ODC1 
                 0.00887527 
                 0.14996152 
                 −0.9667204 
                 0.36602191 
                 212.830324 
                 −2.6411544 
               
               
                 Fbxo11 
                 ODC1 
                 0.01045343 
                 0.16238522 
                 −0.9275589 
                 0.35889412 
                 203.02041 
                 −2.5844919 
               
               
                 Chd8 
                 IN2 
                 0.01054444 
                 0.16238522 
                 0.43024856 
                 0.16587328 
                 134.240663 
                 2.59383876 
               
               
                 Ddx3x 
                 ODC1 
                 0.01088716 
                 0.16238522 
                 −0.9653681 
                 0.37586225 
                 217.228987 
                 −2.5684094 
               
               
                 Dscam 
                 IN2 
                 0.01093615 
                 0.16238522 
                 0.4258342 
                 0.16578804 
                 201.473159 
                 2.56854602 
               
               
                 Qrich1 
                 ODC1 
                 0.01173012 
                 0.16458641 
                 −0.9313837 
                 0.36624599 
                 204.035693 
                 −2.543055 
               
               
                 Chd2 
                 ODC1 
                 0.01175617 
                 0.16458641 
                 −1.0281392 
                 0.40542834 
                 281.292453 
                 −2.5359333 
               
               
                 Tcf7l2 
                 IN1 
                 0.01339322 
                 0.17724723 
                 0.49096525 
                 0.19782395 
                 507.977987 
                 2.48182912 
               
               
                 Gatad2b 
                 PN3 
                 0.0136635 
                 0.17724723 
                 −0.4987271 
                 0.20136181 
                 406.623917 
                 −2.4767708 
               
               
                 Upf3b 
                 IN2 
                 0.01414945 
                 0.17724723 
                 0.36447251 
                 0.14626047 
                 113.504038 
                 2.49194125 
               
               
                 Fbxo11 
                 IN1 
                 0.01458943 
                 0.17724723 
                 0.3383005 
                 0.13719383 
                 183.060255 
                 2.4658579 
               
               
                 Setd2 
                 IN1 
                 0.01510976 
                 0.17724723 
                 0.34155837 
                 0.13932971 
                 194.86755 
                 2.45143958 
               
               
                 Myst4 
                 IN2 
                 0.01515849 
                 0.17724723 
                 0.39493793 
                 0.16148755 
                 247.205754 
                 2.44562457 
               
               
                 Upf3b 
                 PN1 
                 0.01519262 
                 0.17724723 
                 −0.4070623 
                 0.16597076 
                 169.947113 
                 −2.4526144 
               
               
                 Tcf20 
                 IN2 
                 0.0161103 
                 0.18068554 
                 0.36449713 
                 0.14978219 
                 152.321615 
                 2.43351449 
               
               
                 Chd8 
                 PN3 
                 0.01622482 
                 0.18068554 
                 −0.3964885 
                 0.16263137 
                 120.925541 
                 −2.4379583 
               
               
                 Pten 
                 PN1 
                 0.01664414 
                 0.18123622 
                 −0.4317364 
                 0.17778026 
                 120.124443 
                 −2.4284835 
               
               
                 Fbxo11 
                 PN1 
                 0.01737135 
                 0.18504266 
                 −0.3819204 
                 0.15903035 
                 175.220643 
                 −2.4015567 
               
               
                 Myst4 
                 ODC1 
                 0.01836352 
                 0.19144941 
                 −0.9060429 
                 0.38157756 
                 239.451657 
                 −2.374466 
               
               
                 Pogz 
                 PN1 
                 0.02004044 
                 0.19725751 
                 −0.4485723 
                 0.19115805 
                 178.826794 
                 −2.3466043 
               
               
                 Stard9 
                 ODC1 
                 0.020834 
                 0.19725751 
                 −0.8580433 
                 0.3682325 
                 192.429626 
                 −2.3301674 
               
               
                 Pten 
                 PN3 
                 0.02105492 
                 0.19725751 
                 −0.3818055 
                 0.16250732 
                 87.3692069 
                 −2.3494663 
               
               
                 Asxl3 
                 PN1 
                 0.02109219 
                 0.19725751 
                 −0.3682935 
                 0.15803759 
                 152.774857 
                 −2.3304172 
               
               
                 Tnrc6b 
                 ODC1 
                 0.02122581 
                 0.19725751 
                 −0.8052107 
                 0.34670495 
                 197.666663 
                 −2.3224665 
               
               
                 Satb2 
                 PN3 
                 0.02133602 
                 0.19725751 
                 −0.4528999 
                 0.19376672 
                 104.021141 
                 −2.3373463 
               
               
                 Scn2a1 
                 IN1 
                 0.0230174 
                 0.19966279 
                 0.31470458 
                 0.13735074 
                 195.086726 
                 2.2912478 
               
               
                 Fbxo11 
                 IN2 
                 0.02372589 
                 0.19966279 
                 0.33794433 
                 0.14810503 
                 172.194808 
                 2.28178825 
               
               
                 Spen 
                 PN1 
                 0.0238842 
                 0.19966279 
                 −0.4196992 
                 0.18426972 
                 186.285964 
                 −2.2776354 
               
               
                 Spen 
                 PN3 
                 0.02390832 
                 0.19966279 
                 −0.3931063 
                 0.17243082 
                 164.025561 
                 −2.2797916 
               
               
                 Satb2 
                 ODC1 
                 0.02433803 
                 0.19966279 
                 −1.0825085 
                 0.47795154 
                 261.500448 
                 −2.2648918 
               
               
                 Ank2 
                 ODC1 
                 0.02442785 
                 0.19966279 
                 −0.9707684 
                 0.42933031 
                 317.630567 
                 −2.2611224 
               
               
                 Adnp 
                 PN3 
                 0.0244485 
                 0.19966279 
                 −0.343116 
                 0.15053245 
                 117.67566 
                 −2.279349 
               
               
                 Setd5 
                 IN1 
                 0.02532973 
                 0.20346835 
                 0.32065153 
                 0.14233776 
                 205.847053 
                 2.25275093 
               
               
                 Med13l 
                 ODC1 
                 0.02633977 
                 0.20648176 
                 −0.8484613 
                 0.37959847 
                 236.953357 
                 −2.2351548 
               
               
                 Syngap1 
                 ODC1 
                 0.02686339 
                 0.20648176 
                 −0.8432233 
                 0.37830387 
                 212.582942 
                 −2.2289577 
               
               
                 Larp4b 
                 PN3 
                 0.0271183 
                 0.20648176 
                 −0.5524442 
                 0.24962064 
                 979.063936 
                 −2.2131352 
               
               
                 Arid1b 
                 PN1 
                 0.02739044 
                 0.20648176 
                 −0.4848622 
                 0.21887869 
                 349.197284 
                 −2.2152098 
               
               
                 Med13l 
                 PN1 
                 0.02833533 
                 0.21036833 
                 −0.4221952 
                 0.19034411 
                 126.482012 
                 −2.2180628 
               
               
                 Ctnnb1 
                 PN3 
                 0.02930188 
                 0.2142973 
                 −0.3023499 
                 0.13712463 
                 124.112709 
                 −2.2049277 
               
               
                 Kdm5b 
                 PN3 
                 0.03022414 
                 0.21657 
                 −0.3666168 
                 0.16820822 
                 250.250501 
                 −2.1795415 
               
               
                 Setd5 
                 PN1 
                 0.03059615 
                 0.21657 
                 −0.3825701 
                 0.17570488 
                 205.104462 
                 −2.1773448 
               
               
                 Cul3 
                 PN3 
                 0.03110538 
                 0.21657 
                 −0.3222806 
                 0.14670126 
                 75.3611305 
                 −2.1968495 
               
               
                 Mll1 
                 IN1 
                 0.03138055 
                 0.21657 
                 0.31105438 
                 0.14346563 
                 191.562139 
                 2.16814569 
               
               
                 Setd2 
                 PN3 
                 0.03412606 
                 0.22799966 
                 −0.3428662 
                 0.16077114 
                 208.133476 
                 −2.1326353 
               
               
                 Larp4b 
                 IN1 
                 0.03430065 
                 0.22799966 
                 0.37874849 
                 0.17834605 
                 406.716022 
                 2.12367182 
               
               
                 Ddx3x 
                 PN3 
                 0.0344326 
                 0.22799966 
                 −0.3771746 
                 0.17743034 
                 269.461882 
                 −2.1257614 
               
               
                 Syngap1 
                 PN1 
                 0.03640184 
                 0.23782535 
                 −0.3736477 
                 0.17736193 
                 198.239023 
                 −2.106696 
               
               
                 Pten 
                 IN1 
                 0.03802571 
                 0.24297605 
                 0.34718755 
                 0.1665048 
                 261.467272 
                 2.08515043 
               
               
                 Med13l 
                 PN3 
                 0.03818195 
                 0.24297605 
                 −0.3667481 
                 0.17429104 
                 88.8959415 
                 −2.104228 
               
               
                 Asxl3 
                 ODC1 
                 0.04022464 
                 0.24640322 
                 −0.742785 
                 0.35968264 
                 196.5631 
                 −2.0651123 
               
               
                 Tcf20 
                 ODC1 
                 0.04045543 
                 0.24640322 
                 −0.7731876 
                 0.37499938 
                 209.835499 
                 −2.0618369 
               
               
                 Setd2 
                 PN1 
                 0.04115977 
                 0.24640322 
                 −0.3500251 
                 0.17044045 
                 226.158301 
                 −2.0536507 
               
               
                 Dscam 
                 ODC1 
                 0.04130123 
                 0.24640322 
                 −0.7904061 
                 0.385021 
                 213.722084 
                 −2.052891 
               
               
                 Mbd5 
                 IN2 
                 0.04174905 
                 0.24640322 
                 0.30979166 
                 0.15103144 
                 174.007298 
                 2.0511734 
               
               
                 Ank2 
                 PN3 
                 0.04204699 
                 0.24640322 
                 −0.3842745 
                 0.18749879 
                 160.123748 
                 −2.0494773 
               
               
                 Larp4b 
                 ODC1 
                 0.04329398 
                 0.24640322 
                 −0.8450975 
                 0.41615916 
                 262.141864 
                 −2.0307073 
               
               
                 Cul3 
                 IN1 
                 0.04334757 
                 0.24640322 
                 0.2939761 
                 0.14459491 
                 202.462117 
                 2.03310131 
               
               
                 Mbd5 
                 PN3 
                 0.04337062 
                 0.24640322 
                 −0.3026135 
                 0.14832803 
                 129.280204 
                 −2.0401637 
               
               
                 Spen 
                 ODC1 
                 0.04386743 
                 0.24640322 
                 −0.7421949 
                 0.36617305 
                 222.205812 
                 −2.0268966 
               
               
                 Chd2 
                 PN1 
                 0.04425201 
                 0.24640322 
                 −0.4768338 
                 0.23572346 
                 228.742435 
                 −2.0228524 
               
               
                 Dscam 
                 PN3 
                 0.04793083 
                 0.26388884 
                 −0.3410084 
                 0.17074224 
                 127.520632 
                 −1.9972118 
               
               
                 Tnrc6b 
                 IN2 
                 0.04879687 
                 0.26567187 
                 0.27910155 
                 0.14062215 
                 168.207503 
                 1.9847624 
               
               
                 Qrich1 
                 IN1 
                 0.04935811 
                 0.26577441 
                 0.27966809 
                 0.14139027 
                 192.337987 
                 1.97798673 
               
               
                 Setd2 
                 IN2 
                 0.05033431 
                 0.26754815 
                 0.29806063 
                 0.15124956 
                 175.698077 
                 1.9706545 
               
               
                 Wac 
                 IN1 
                 0.05089802 
                 0.26754815 
                 0.27734145 
                 0.14118309 
                 195.732876 
                 1.9644098 
               
               
                 Larp4b 
                 PN6 
                 0.05132556 
                 0.26754815 
                 0.50145802 
                 0.25690778 
                 742.902409 
                 1.95189892 
               
               
                 Ddx3x 
                 PN1 
                 0.05237953 
                 0.26839548 
                 −0.3634946 
                 0.18658399 
                 284.500854 
                 −1.9481556 
               
               
                 Mbd5 
                 PN1 
                 0.0525836 
                 0.26839548 
                 −0.3123583 
                 0.15990287 
                 153.826324 
                 −1.9534253 
               
               
                 Qrich1 
                 IN2 
                 0.05350982 
                 0.26877191 
                 0.29827949 
                 0.15346176 
                 178.217697 
                 1.94367306 
               
               
                 Wac 
                 IN2 
                 0.05375438 
                 0.26877191 
                 0.29903955 
                 0.15408196 
                 191.287276 
                 1.94078235 
               
               
                 Tcf20 
                 IN1 
                 0.0560518 
                 0.27742809 
                 0.26772604 
                 0.13926607 
                 189.81 
                 1.92240682 
               
               
                 Satb2 
                 IN1 
                 0.05704571 
                 0.27952395 
                 0.33435664 
                 0.17448217 
                 166.202049 
                 1.9162797 
               
               
                 Pogz 
                 ODC1 
                 0.05839363 
                 0.28329584 
                 −0.7904375 
                 0.41609539 
                 314.322084 
                 −1.8996545 
               
               
                 Syngap1 
                 IN1 
                 0.05910231 
                 0.28392285 
                 0.2917968 
                 0.15382473 
                 227.453888 
                 1.89694341 
               
               
                 Spen 
                 IN2 
                 0.06475702 
                 0.30563812 
                 0.31586545 
                 0.17012955 
                 211.386758 
                 1.8566172 
               
               
                 Larp4b 
                 PN1 
                 0.06487013 
                 0.30563812 
                 −0.4707516 
                 0.25468242 
                 911.604736 
                 −1.8483866 
               
               
                 Myst4 
                 IN1 
                 0.0655383 
                 0.30584539 
                 0.26702444 
                 0.14430914 
                 230.948808 
                 1.850364 
               
               
                 Tcf20 
                 PN3 
                 0.06653463 
                 0.30756576 
                 −0.2880181 
                 0.15571213 
                 135.70089 
                 −1.8496831 
               
               
                 Setd5 
                 IN2 
                 0.06864764 
                 0.3143677 
                 0.28647332 
                 0.15651066 
                 204.734493 
                 1.8303758 
               
               
                 Mbd5 
                 IN1 
                 0.07279319 
                 0.3302654 
                 0.25212592 
                 0.1397305 
                 185.704142 
                 1.80437291 
               
               
                 Ctnnb1 
                 IN2 
                 0.07694194 
                 0.34405266 
                 0.25215923 
                 0.14156716 
                 147.221051 
                 1.78119869 
               
               
                 Wac 
                 PN3 
                 0.07723631 
                 0.34405266 
                 −0.2810658 
                 0.15806527 
                 163.442956 
                 −1.778163 
               
               
                 Pten 
                 ODC1 
                 0.07912183 
                 0.34927652 
                 −0.7234123 
                 0.41032923 
                 249.921845 
                 −1.7630046 
               
               
                 Chd8 
                 IN1 
                 0.08190038 
                 0.35831416 
                 0.27098887 
                 0.15495404 
                 194.118724 
                 1.74883382 
               
               
                 Dyrk1a 
                 ODC1 
                 0.08457922 
                 0.36675946 
                 −0.6877839 
                 0.39675887 
                 195.89186 
                 −1.7335061 
               
               
                 Arid1b 
                 PN3 
                 0.08847035 
                 0.37446275 
                 −0.3579273 
                 0.20950736 
                 340.051858 
                 −1.7084233 
               
               
                 Ctnnb1 
                 IN1 
                 0.08867228 
                 0.37446275 
                 0.22821284 
                 0.13326931 
                 167.304807 
                 1.71241854 
               
               
                 Tnrc6b 
                 IN1 
                 0.09036519 
                 0.37446275 
                 0.22254693 
                 0.130697 
                 177.13054 
                 1.70277002 
               
               
                 Scn2a1 
                 Astro1 
                 0.09069609 
                 0.37446275 
                 0.36728677 
                 0.21628943 
                 256.616648 
                 1.69812627 
               
               
                 Tcf7l2 
                 ODC1 
                 0.09112832 
                 0.37446275 
                 −0.7269746 
                 0.42898237 
                 315.640752 
                 −1.6946491 
               
               
                 Adnp 
                 IN2 
                 0.09120538 
                 0.37446275 
                 0.29642386 
                 0.17484412 
                 259.731956 
                 1.69536076 
               
               
                 Mll1 
                 Astro2 
                 0.09229944 
                 0.37446275 
                 −0.425959 
                 0.25202661 
                 240.202703 
                 −1.6901349 
               
               
                 Spen 
                 IN1 
                 0.09246937 
                 0.37446275 
                 0.25992735 
                 0.15381146 
                 218.837373 
                 1.68990891 
               
               
                 Gatad2b 
                 PN1 
                 0.09387819 
                 0.37550282 
                 −0.3517088 
                 0.2094425 
                 401.927081 
                 −1.6792618 
               
               
                 Mll1 
                 PN1 
                 0.09488777 
                 0.37550282 
                 −0.2709321 
                 0.16121128 
                 152.462455 
                 −1.6806027 
               
               
                 Pten 
                 IN2 
                 0.09516184 
                 0.37550282 
                 0.31611869 
                 0.1887937 
                 281.031211 
                 1.67441336 
               
               
                 Cul3 
                 IN2 
                 0.09579154 
                 0.37550282 
                 0.262326 
                 0.15651377 
                 151.35227 
                 1.676057 
               
               
                 Gatad2b 
                 PN6 
                 0.09829932 
                 0.37751721 
                 0.35363523 
                 0.21330513 
                 327.153359 
                 1.6578843 
               
               
                 Chd8 
                 PN1 
                 0.0983165 
                 0.37751721 
                 −0.2925584 
                 0.17583348 
                 144.199437 
                 −1.6638381 
               
               
                 Satb2 
                 IN2 
                 0.09861674 
                 0.37751721 
                 0.29580301 
                 0.17680564 
                 72.7126278 
                 1.67304062 
               
               
                 Chd2 
                 IN2 
                 0.09965973 
                 0.37855246 
                 0.44078116 
                 0.26747441 
                 1067.50326 
                 1.6479377 
               
               
                 Wac 
                 PN1 
                 0.10168008 
                 0.38325568 
                 −0.2777584 
                 0.16887424 
                 189.056758 
                 −1.6447645 
               
               
                 Arid1b 
                 IN2 
                 0.10301147 
                 0.38531008 
                 0.28408311 
                 0.17369228 
                 291.711235 
                 1.63555399 
               
               
                 Arid1b 
                 ODC1 
                 0.10732648 
                 0.39840892 
                 −0.6707221 
                 0.41541466 
                 340.657567 
                 −1.6145846 
               
               
                 Ank2 
                 PN2 
                 0.10908119 
                 0.40187805 
                 −0.4488767 
                 0.2792997 
                 297.746012 
                 −1.6071508 
               
               
                 Larp4b 
                 Astro2 
                 0.11160355 
                 0.40810254 
                 −0.5305859 
                 0.33297246 
                 572.812177 
                 −1.5934827 
               
               
                 Dyrk1a 
                 PN4 
                 0.11450881 
                 0.41562458 
                 −0.3197754 
                 0.20166014 
                 185.336932 
                 −1.5857144 
               
               
                 Kdm5b 
                 IN1 
                 0.11580458 
                 0.41723709 
                 0.23104577 
                 0.14623668 
                 187.565094 
                 1.57994405 
               
               
                 Scn2a1 
                 ODC1 
                 0.11710172 
                 0.41883099 
                 −0.5656577 
                 0.35946557 
                 207.494277 
                 −1.5736076 
               
               
                 Mll1 
                 ODC1 
                 0.11860514 
                 0.4211342 
                 −0.5759361 
                 0.36747017 
                 202.58449 
                 −1.5673004 
               
               
                 Ash1l 
                 IN2 
                 0.12198562 
                 0.4295698 
                 0.24484961 
                 0.15774639 
                 231.723531 
                 1.55217257 
               
               
                 Gatad2b 
                 PN2 
                 0.12273423 
                 0.4295698 
                 −0.408516 
                 0.26417443 
                 437.390081 
                 −1.5463874 
               
               
                 Tcf7l2 
                 PN2 
                 0.12414421 
                 0.43142314 
                 −0.4964536 
                 0.32236115 
                 529.78759 
                 −1.5400541 
               
               
                 Satb2 
                 PN1 
                 0.12763907 
                 0.4402084 
                 −0.3223574 
                 0.21033944 
                 140.145731 
                 −1.5325581 
               
               
                 Tcf20 
                 PN1 
                 0.12846898 
                 0.4402084 
                 −0.2559473 
                 0.16750743 
                 161.972018 
                 −1.527976 
               
               
                 Mll1 
                 IN2 
                 0.13081046 
                 0.44511892 
                 0.23098876 
                 0.15171294 
                 107.502688 
                 1.52253831 
               
               
                 Tnrc6b 
                 PN1 
                 0.13535824 
                 0.45441243 
                 −0.2179682 
                 0.14526049 
                 167.56762 
                 −1.500533 
               
               
                 Gatad2b 
                 Astro1 
                 0.13539636 
                 0.45441243 
                 0.35804573 
                 0.23928536 
                 382.716897 
                 1.49631272 
               
               
                 Tcf7l2 
                 IN2 
                 0.14036834 
                 0.46785855 
                 0.35824741 
                 0.24273829 
                 814.431287 
                 1.47585866 
               
               
                 Tcf7l2 
                 PN4 
                 0.14168645 
                 0.46785855 
                 0.40320111 
                 0.27385936 
                 422.459172 
                 1.47229261 
               
               
                 Upf3b 
                 IN1 
                 0.14226719 
                 0.46785855 
                 0.20435692 
                 0.13864556 
                 177.358286 
                 1.47395212 
               
               
                 Ddx3x 
                 IN1 
                 0.14617494 
                 0.47750481 
                 0.22821809 
                 0.15665778 
                 313.248231 
                 1.45679382 
               
               
                 Ank2 
                 Astro1 
                 0.14781237 
                 0.47965605 
                 0.39956277 
                 0.27564342 
                 495.801747 
                 1.44956397 
               
               
                 Arid1b 
                 PN5 
                 0.15332592 
                 0.49427435 
                 0.42590913 
                 0.29779043 
                 462.49406 
                 1.43023109 
               
               
                 Mll1 
                 PN3 
                 0.15854256 
                 0.50487174 
                 −0.2117959 
                 0.14926807 
                 119.024155 
                 −1.4188965 
               
               
                 Mll1 
                 Astro1 
                 0.15867398 
                 0.50487174 
                 0.31307698 
                 0.22144227 
                 247.642878 
                 1.41380856 
               
               
                 Setd2 
                 PN2 
                 0.16024002 
                 0.50656522 
                 −0.3222111 
                 0.22894474 
                 336.199893 
                 −1.4073752 
               
               
                 Spen 
                 Astro2 
                 0.16305787 
                 0.51216895 
                 −0.3612308 
                 0.25814225 
                 229.672039 
                 −1.3993477 
               
               
                 Chd2 
                 PN5 
                 0.17046605 
                 0.5320278 
                 0.4730862 
                 0.34454298 
                 416.031804 
                 1.37308327 
               
               
                 Dyrk1a 
                 PN1 
                 0.17268329 
                 0.53285394 
                 −0.2413247 
                 0.17599866 
                 129.993509 
                 −1.3711736 
               
               
                 Gatad2b 
                 IN1 
                 0.17290567 
                 0.53285394 
                 0.22695208 
                 0.16611379 
                 294.16276 
                 1.36624469 
               
               
                 Pogz 
                 PN4 
                 0.17530734 
                 0.53595644 
                 −0.2911766 
                 0.21412822 
                 215.406937 
                 −1.3598236 
               
               
                 Scn2a1 
                 IN2 
                 0.17655489 
                 0.53595644 
                 0.2022653 
                 0.14904597 
                 170.403836 
                 1.35706653 
               
               
                 Tnrc6b 
                 PN3 
                 0.17827442 
                 0.53595644 
                 −0.1830557 
                 0.13533739 
                 146.286231 
                 −1.3525876 
               
               
                 Mbd5 
                 PN4 
                 0.17828755 
                 0.53595644 
                 −0.2444733 
                 0.18098711 
                 200.732389 
                 −1.3507771 
               
               
                 Spen 
                 PN6 
                 0.18015818 
                 0.53760242 
                 0.25570565 
                 0.18995218 
                 159.791863 
                 1.34615799 
               
               
                 Setd2 
                 Mg1 
                 0.18102938 
                 0.53760242 
                 0.33240861 
                 0.24760798 
                 191.556783 
                 1.34247939 
               
               
                 Dyrk1a 
                 PN3 
                 0.19094242 
                 0.5636252 
                 −0.2129695 
                 0.16175276 
                 100.934316 
                 −1.3166361 
               
               
                 Adnp 
                 IN1 
                 0.19405063 
                 0.56937013 
                 0.20259153 
                 0.15556103 
                 240.369577 
                 1.30232834 
               
               
                 Stard9 
                 IN2 
                 0.19661319 
                 0.57102142 
                 0.1876929 
                 0.14470956 
                 149.890689 
                 1.29703178 
               
               
                 Pogz 
                 Astro2 
                 0.19787668 
                 0.57102142 
                 −0.3658267 
                 0.28362491 
                 389.407962 
                 −1.2898256 
               
               
                 Larp4b 
                 Astro3 
                 0.19810947 
                 0.57102142 
                 −0.3927733 
                 0.30489005 
                 663.604701 
                 −1.2882456 
               
               
                 Cul3 
                 PN2 
                 0.20144859 
                 0.57725033 
                 −0.2981748 
                 0.23287074 
                 281.30109 
                 −1.2804304 
               
               
                 Scn2a1 
                 Astro2 
                 0.20428102 
                 0.58030982 
                 −0.313115 
                 0.24598862 
                 241.920131 
                 −1.2728842 
               
               
                 Asxl3 
                 IN1 
                 0.20776789 
                 0.58030982 
                 0.17703497 
                 0.14002279 
                 177.659476 
                 1.26432973 
               
               
                 Spen 
                 PN5 
                 0.2082352 
                 0.58030982 
                 0.35063143 
                 0.27814934 
                 378.030407 
                 1.26058696 
               
               
                 Asxl3 
                 PN5 
                 0.2099462 
                 0.58030982 
                 0.31132952 
                 0.24785403 
                 338.36755 
                 1.25610026 
               
               
                 Wac 
                 ODC1 
                 0.21020503 
                 0.58030982 
                 −0.4557558 
                 0.36261097 
                 208.64131 
                 −1.2568726 
               
               
                 Adnp 
                 Astro2 
                 0.21063655 
                 0.58030982 
                 −0.3244788 
                 0.25862885 
                 288.123197 
                 −1.2546116 
               
               
                 Med13l 
                 IN1 
                 0.21080643 
                 0.58030982 
                 0.18733757 
                 0.14928667 
                 227.813488 
                 1.25488472 
               
               
                 Chd2 
                 PN2 
                 0.22092133 
                 0.6020548 
                 −0.3866106 
                 0.31528882 
                 359.762296 
                 −1.2262108 
               
               
                 Tcf20 
                 PN6 
                 0.22258013 
                 0.6020548 
                 0.21208773 
                 0.17312114 
                 141.449581 
                 1.22508282 
               
               
                 Arid1b 
                 PN4 
                 0.22302392 
                 0.6020548 
                 −0.2848739 
                 0.23337412 
                 353.029407 
                 −1.2206746 
               
               
                 Ctnnb1 
                 PN2 
                 0.22362035 
                 0.6020548 
                 −0.2589992 
                 0.21231357 
                 258.045902 
                 −1.2198899 
               
               
                 Tcf7l2 
                 PN6 
                 0.22538342 
                 0.60348565 
                 0.32174365 
                 0.26492906 
                 354.528615 
                 1.21445211 
               
               
                 Fbxo11 
                 PN6 
                 0.22927125 
                 0.6105593 
                 0.1981227 
                 0.16412604 
                 150.640324 
                 1.2071375 
               
               
                 Dscam 
                 PN5 
                 0.23567737 
                 0.62422655 
                 0.3388436 
                 0.2852651 
                 364.675847 
                 1.18782003 
               
               
                 Myst4 
                 Mg1 
                 0.23944631 
                 0.63079943 
                 0.2880371 
                 0.24401789 
                 174.937035 
                 1.18039337 
               
               
                 Ash1l 
                 PN6 
                 0.24139533 
                 0.63253321 
                 0.19213456 
                 0.16331081 
                 139.848033 
                 1.17649626 
               
               
                 Kdm5b 
                 Mg2 
                 0.24812997 
                 0.64331493 
                 0.29832927 
                 0.25769887 
                 244.491088 
                 1.15766619 
               
               
                 Stard9 
                 PN2 
                 0.24813576 
                 0.64331493 
                 −0.2539718 
                 0.2194641 
                 287.680254 
                 −1.1572364 
               
               
                 Satb2 
                 PN6 
                 0.25535028 
                 0.65853493 
                 0.24898471 
                 0.21792608 
                 129.252133 
                 1.14251914 
               
               
                 Myst4 
                 PN3 
                 0.2591674 
                 0.66135943 
                 −0.1766618 
                 0.15613455 
                 206.747668 
                 −1.1314715 
               
               
                 Stard9 
                 IN1 
                 0.26111036 
                 0.66135943 
                 0.1526452 
                 0.13540113 
                 177.913794 
                 1.12735543 
               
               
                 Upf3b 
                 Astro1 
                 0.26418265 
                 0.66135943 
                 0.2334001 
                 0.2085591 
                 246.693216 
                 1.11910773 
               
               
                 Larp4b 
                 PN4 
                 0.26424021 
                 0.66135943 
                 0.28779177 
                 0.25757705 
                 714.248177 
                 1.11730363 
               
               
                 Pogz 
                 IN1 
                 0.26468335 
                 0.66135943 
                 0.17946308 
                 0.16058219 
                 286.598732 
                 1.1175777 
               
               
                 Cul3 
                 Astro1 
                 0.26499079 
                 0.66135943 
                 0.23993624 
                 0.2148091 
                 272.058753 
                 1.11697427 
               
               
                 Chd8 
                 Astro1 
                 0.266346 
                 0.66135943 
                 0.27128152 
                 0.24371321 
                 388.536817 
                 1.11311783 
               
               
                 Chd2 
                 PN3 
                 0.2672432 
                 0.66135943 
                 −0.2475013 
                 0.22250899 
                 214.888311 
                 −1.1123205 
               
               
                 Dscam 
                 PN1 
                 0.26864254 
                 0.66148163 
                 −0.2043485 
                 0.18407066 
                 155.491677 
                 −1.1101633 
               
               
                 Setd5 
                 Astro1 
                 0.27564986 
                 0.66902663 
                 0.23497015 
                 0.21510798 
                 272.623952 
                 1.09233583 
               
               
                 Pogz 
                 Astro1 
                 0.27917651 
                 0.66902663 
                 0.27448439 
                 0.25332964 
                 440.294827 
                 1.08350681 
               
               
                 Adnp 
                 PN5 
                 0.28088284 
                 0.66902663 
                 0.27805976 
                 0.25746749 
                 355.667461 
                 1.0799801 
               
               
                 Med13l 
                 PN4 
                 0.28150917 
                 0.66902663 
                 −0.233994 
                 0.2167198 
                 210.305599 
                 −1.0797075 
               
               
                 Chd8 
                 Astro3 
                 0.28292114 
                 0.66902663 
                 −0.2694012 
                 0.25056023 
                 409.011186 
                 −1.0751953 
               
               
                 Stard9 
                 PN6 
                 0.28442829 
                 0.66902663 
                 0.17247437 
                 0.16040737 
                 120.103158 
                 1.07522723 
               
               
                 Larp4b 
                 Astro1 
                 0.28457803 
                 0.66902663 
                 0.32182506 
                 0.30050641 
                 675.092649 
                 1.07094243 
               
               
                 Arid1b 
                 IN1 
                 0.28621596 
                 0.66902663 
                 0.16376974 
                 0.15323487 
                 249.34702 
                 1.06874984 
               
               
                 Wac 
                 Astro3 
                 0.28742399 
                 0.66902663 
                 0.23149657 
                 0.21715918 
                 254.435602 
                 1.06602248 
               
               
                 Spen 
                 Mg1 
                 0.28953893 
                 0.66902663 
                 0.26375857 
                 0.24826578 
                 172.318778 
                 1.06240404 
               
               
                 Syngap1 
                 Astro3 
                 0.2896817 
                 0.66902663 
                 0.25161088 
                 0.2372046 
                 293.96531 
                 1.06073359 
               
               
                 Chd2 
                 Astro1 
                 0.2909498 
                 0.66902663 
                 0.315637 
                 0.298662 
                 701.645834 
                 1.05683682 
               
               
                 Ank2 
                 Mg2 
                 0.29118172 
                 0.66902663 
                 −0.3375746 
                 0.31927558 
                 313.72542 
                 −1.0573141 
               
               
                 Chd8 
                 Astro2 
                 0.29237098 
                 0.66902663 
                 −0.2886983 
                 0.27376993 
                 348.684426 
                 −1.0545289 
               
               
                 Gatad2b 
                 IN2 
                 0.2928021 
                 0.66902663 
                 0.20273642 
                 0.19244871 
                 379.384305 
                 1.05345689 
               
               
                 Qrich1 
                 PN1 
                 0.29449286 
                 0.66902663 
                 −0.1798966 
                 0.17107267 
                 169.072756 
                 −1.0515802 
               
               
                 Ash1l 
                 Mg1 
                 0.29599175 
                 0.66902663 
                 0.24037313 
                 0.2292543 
                 159.773867 
                 1.04850001 
               
               
                 Scn2a1 
                 PN2 
                 0.29628322 
                 0.66902663 
                 −0.2415428 
                 0.23089106 
                 322.398671 
                 −1.0461332 
               
               
                 Qrich1 
                 PN3 
                 0.30382266 
                 0.68290414 
                 −0.164423 
                 0.15932412 
                 142.200743 
                 −1.0320031 
               
               
                 Upf3b 
                 Astro2 
                 0.3100974 
                 0.69382524 
                 −0.2415959 
                 0.23750378 
                 233.429045 
                 −1.0172295 
               
               
                 Ank2 
                 Astro2 
                 0.31487322 
                 0.70130855 
                 −0.3096623 
                 0.30774306 
                 425.796538 
                 −1.0062366 
               
               
                 Ank2 
                 IN2 
                 0.31867065 
                 0.70487084 
                 0.23280309 
                 0.23330094 
                 739.66078 
                 0.99786605 
               
               
                 Pten 
                 PN6 
                 0.31934965 
                 0.70487084 
                 0.18489405 
                 0.18483706 
                 110.356334 
                 1.00030833 
               
               
                 Ash1l 
                 PN5 
                 0.32164727 
                 0.70675857 
                 0.24582022 
                 0.24766693 
                 335.719445 
                 0.99254357 
               
               
                 Kdm5b 
                 PN6 
                 0.32516408 
                 0.70888746 
                 0.17913211 
                 0.18165818 
                 221.218852 
                 0.98609438 
               
               
                 Pten 
                 PN5 
                 0.32550955 
                 0.70888746 
                 0.27620477 
                 0.2805569 
                 375.622717 
                 0.98448755 
               
               
                 Wac 
                 PN2 
                 0.3270718 
                 0.70913798 
                 −0.2258634 
                 0.23012409 
                 330.278873 
                 −0.9814855 
               
               
                 Asxl3 
                 PN4 
                 0.32996088 
                 0.7098563 
                 −0.1743863 
                 0.17858207 
                 205.743627 
                 −0.9765053 
               
               
                 Setd5 
                 PN6 
                 0.33139571 
                 0.7098563 
                 0.17612834 
                 0.18082253 
                 173.133399 
                 0.97403978 
               
               
                 Mll1 
                 Mg1 
                 0.33174917 
                 0.7098563 
                 0.2374933 
                 0.24400752 
                 174.593202 
                 0.9733032 
               
               
                 Gatad2b 
                 Mg1 
                 0.33749307 
                 0.71900697 
                 0.28009141 
                 0.29150708 
                 269.895696 
                 0.96083911 
               
               
                 Ctnnb1 
                 Astro2 
                 0.34067901 
                 0.72265244 
                 −0.2206167 
                 0.23101178 
                 209.17035 
                 −0.9550021 
               
               
                 Upf3b 
                 Astro3 
                 0.34842511 
                 0.73345953 
                 −0.2040676 
                 0.21726634 
                 275.37402 
                 −0.9392508 
               
               
                 Upf3b 
                 PN6 
                 0.34876749 
                 0.73345953 
                 0.16104828 
                 0.17134306 
                 150.369691 
                 0.93991712 
               
               
                 Syngap1 
                 PN5 
                 0.3504851 
                 0.73352679 
                 0.24885976 
                 0.26622046 
                 383.858061 
                 0.93478827 
               
               
                 Chd8 
                 PN5 
                 0.35179346 
                 0.73352679 
                 0.25637813 
                 0.27500442 
                 377.407565 
                 0.93226913 
               
               
                 Syngap1 
                 IN2 
                 0.36003978 
                 0.74566045 
                 0.15707866 
                 0.1712751 
                 231.007428 
                 0.91711322 
               
               
                 Pogz 
                 PN3 
                 0.36065618 
                 0.74566045 
                 −0.1637614 
                 0.1786314 
                 159.318334 
                 −0.9167559 
               
               
                 Fbxo11 
                 PN5 
                 0.36688302 
                 0.75394336 
                 0.22246052 
                 0.24621665 
                 344.919541 
                 0.90351536 
               
               
                 Dscam 
                 Mg1 
                 0.3681958 
                 0.75394336 
                 0.21813876 
                 0.24171844 
                 157.528927 
                 0.90244979 
               
               
                 Scn2a1 
                 PN4 
                 0.36967906 
                 0.75394336 
                 −0.1688266 
                 0.18781037 
                 219.688342 
                 −0.8989208 
               
               
                 Stard9 
                 PN4 
                 0.37105198 
                 0.75394336 
                 −0.1583032 
                 0.17655611 
                 191.003632 
                 −0.896617 
               
               
                 Fbxo11 
                 Astro1 
                 0.3723557 
                 0.75394336 
                 0.18327708 
                 0.20507238 
                 243.23343 
                 0.89371897 
               
               
                 Qrich1 
                 PN6 
                 0.37555445 
                 0.75422956 
                 0.15701344 
                 0.17665595 
                 147.333811 
                 0.8888092 
               
               
                 Gatad2b 
                 Astro2 
                 0.3769287 
                 0.75422956 
                 −0.2379749 
                 0.26897894 
                 338.090072 
                 −0.8847343 
               
               
                 Pten 
                 PN2 
                 0.37711478 
                 0.75422956 
                 0.22351457 
                 0.25270279 
                 309.686461 
                 0.88449586 
               
               
                 Fbxo11 
                 Mg2 
                 0.38077651 
                 0.75600862 
                 0.20595347 
                 0.23447822 
                 206.348898 
                 0.87834796 
               
               
                 Qrich1 
                 Mg1 
                 0.38109006 
                 0.75600862 
                 0.2023028 
                 0.23023136 
                 138.4375 
                 0.8786935 
               
               
                 Mbd5 
                 Mg1 
                 0.38452002 
                 0.75973713 
                 −0.2074172 
                 0.2378585 
                 158.109345 
                 −0.8720195 
               
               
                 Adnp 
                 PN6 
                 0.3916382 
                 0.77069365 
                 0.14507442 
                 0.16876601 
                 125.508836 
                 0.8596187 
               
               
                 Ddx3x 
                 PN6 
                 0.39395529 
                 0.7710519 
                 0.16308745 
                 0.19096435 
                 237.217842 
                 0.85402038 
               
               
                 Upf3b 
                 Mg1 
                 0.3949674 
                 0.7710519 
                 0.19117885 
                 0.22406111 
                 141.355208 
                 0.85324421 
               
               
                 Chd8 
                 Mg2 
                 0.40271732 
                 0.77865747 
                 0.23435033 
                 0.27957509 
                 244.112695 
                 0.83823752 
               
               
                 Kdm5b 
                 IN2 
                 0.40425802 
                 0.77865747 
                 0.13210385 
                 0.15799775 
                 171.084591 
                 0.83611222 
               
               
                 Larp4b 
                 IN2 
                 0.40506004 
                 0.77865747 
                 0.17765399 
                 0.21319973 
                 537.138806 
                 0.83327496 
               
               
                 Gatad2b 
                 Astro3 
                 0.40579256 
                 0.77865747 
                 −0.2048621 
                 0.2461673 
                 397.05413 
                 −0.8322066 
               
               
                 Arid1b 
                 Mg2 
                 0.40820688 
                 0.77865747 
                 0.23667804 
                 0.2858657 
                 395.568839 
                 0.82793439 
               
               
                 Qrich1 
                 Astro1 
                 0.4087239 
                 0.77865747 
                 0.17267531 
                 0.20866348 
                 250.628231 
                 0.8275301 
               
               
                 Pogz 
                 Astro3 
                 0.40998699 
                 0.77865747 
                 −0.2140939 
                 0.25960974 
                 455.803816 
                 −0.8246759 
               
               
                 Chd2 
                 Mg1 
                 0.41638789 
                 0.78638047 
                 0.2966353 
                 0.36477984 
                 694.380562 
                 0.81318995 
               
               
                 Dyrk1a 
                 PN2 
                 0.41757393 
                 0.78638047 
                 −0.2038823 
                 0.25114365 
                 285.450498 
                 −0.8118155 
               
               
                 Wac 
                 Astro2 
                 0.42177404 
                 0.78638047 
                 −0.1910887 
                 0.23741148 
                 215.430757 
                 −0.804884 
               
               
                 Mll1 
                 PN2 
                 0.42187371 
                 0.78638047 
                 −0.1806247 
                 0.22458766 
                 307.494523 
                 −0.8042504 
               
               
                 Qrich1 
                 PN2 
                 0.42207768 
                 0.78638047 
                 −0.1896844 
                 0.23596813 
                 319.577823 
                 −0.803856 
               
               
                 Tcf20 
                 PN2 
                 0.42484464 
                 0.78804349 
                 −0.1854949 
                 0.23213788 
                 318.195808 
                 −0.7990721 
               
               
                 Mbd5 
                 PN2 
                 0.4274668 
                 0.78804349 
                 −0.1781251 
                 0.22416408 
                 298.318264 
                 −0.7946191 
               
               
                 Kdm5b 
                 PN2 
                 0.42897056 
                 0.78804349 
                 −0.1849706 
                 0.23359622 
                 363.759655 
                 −0.7918391 
               
               
                 Ash1l 
                 IN1 
                 0.43171796 
                 0.78804349 
                 0.11173583 
                 0.1418511 
                 220.84089 
                 0.78769805 
               
               
                 Tcf7l2 
                 Mg2 
                 0.43473347 
                 0.78804349 
                 −0.2276547 
                 0.29106532 
                 306.970019 
                 −0.7821429 
               
               
                 Tnrc6b 
                 PN4 
                 0.43546956 
                 0.78804349 
                 −0.1279096 
                 0.16368773 
                 201.622095 
                 −0.7814243 
               
               
                 Syngap1 
                 PN4 
                 0.43552485 
                 0.78804349 
                 −0.1538676 
                 0.19698378 
                 233.295681 
                 −0.7811183 
               
               
                 Setd2 
                 Astro3 
                 0.43855288 
                 0.78804349 
                 0.17318876 
                 0.22328359 
                 308.525557 
                 0.77564479 
               
               
                 Med13l 
                 Mg2 
                 0.43970721 
                 0.78804349 
                 0.20049065 
                 0.25900987 
                 224.263012 
                 0.77406567 
               
               
                 Ash1l 
                 PN2 
                 0.44039713 
                 0.78804349 
                 −0.1702654 
                 0.22039076 
                 296.653756 
                 −0.7725613 
               
               
                 Arid1b 
                 Astro3 
                 0.44279077 
                 0.78804349 
                 0.20396356 
                 0.26557561 
                 590.635292 
                 0.76800563 
               
               
                 Setd2 
                 Mg2 
                 0.44381775 
                 0.78804349 
                 −0.1968112 
                 0.25660764 
                 251.124337 
                 −0.7669735 
               
               
                 Chd2 
                 PN6 
                 0.44387756 
                 0.78804349 
                 0.18577643 
                 0.24213259 
                 191.451237 
                 0.76725082 
               
               
                 Scn2a1 
                 Mg1 
                 0.44736969 
                 0.79137598 
                 0.18331282 
                 0.24070888 
                 171.909943 
                 0.76155402 
               
               
                 Cul3 
                 Astro2 
                 0.44945353 
                 0.79220227 
                 −0.1846654 
                 0.24378935 
                 258.17649 
                 −0.7574793 
               
               
                 Chd2 
                 Mg2 
                 0.45160892 
                 0.79314829 
                 −0.272229 
                 0.3614571 
                 723.161063 
                 −0.7531433 
               
               
                 Stard9 
                 Astro1 
                 0.45407697 
                 0.79463469 
                 0.15366866 
                 0.20491197 
                 226.792301 
                 0.74992524 
               
               
                 Chd2 
                 PN4 
                 0.45673308 
                 0.79643846 
                 0.19315679 
                 0.25914134 
                 255.017912 
                 0.74537235 
               
               
                 Adnp 
                 Mg2 
                 0.4627929 
                 0.80145772 
                 −0.1843535 
                 0.25067379 
                 240.528335 
                 −0.7354321 
               
               
                 Ddx3x 
                 Astro1 
                 0.46288273 
                 0.80145772 
                 0.17050738 
                 0.23190372 
                 247.57752 
                 0.73525073 
               
               
                 Mll1 
                 PN4 
                 0.47062201 
                 0.81198868 
                 −0.131518 
                 0.18196208 
                 209.737801 
                 −0.7227771 
               
               
                 Chd8 
                 PN6 
                 0.47398974 
                 0.81492973 
                 0.13082562 
                 0.18216889 
                 126.102563 
                 0.71815567 
               
               
                 Ddx3x 
                 Mg1 
                 0.48148878 
                 0.82492833 
                 0.17117616 
                 0.2425225 
                 138.448024 
                 0.70581559 
               
               
                 Spen 
                 Astro1 
                 0.48552364 
                 0.82894279 
                 0.15823059 
                 0.22652187 
                 241.157311 
                 0.69852235 
               
               
                 Med13l 
                 Astro3 
                 0.49403453 
                 0.83898255 
                 0.16628257 
                 0.24283471 
                 295.490422 
                 0.68475619 
               
               
                 Mbd5 
                 Astro1 
                 0.49482849 
                 0.83898255 
                 0.14410931 
                 0.2107764 
                 236.859129 
                 0.68370704 
               
               
                 Syngap1 
                 Mg1 
                 0.50062396 
                 0.84303519 
                 −0.1662291 
                 0.24620909 
                 149.014715 
                 −0.6751544 
               
               
                 Ank2 
                 PN6 
                 0.50065967 
                 0.84303519 
                 0.13985675 
                 0.20716272 
                 148.316286 
                 0.67510576 
               
               
                 Scn2a1 
                 PN5 
                 0.50329224 
                 0.84456574 
                 0.17164133 
                 0.25618965 
                 368.015776 
                 0.66997759 
               
               
                 Satb2 
                 Astro3 
                 0.50632628 
                 0.84675727 
                 −0.1923981 
                 0.28920351 
                 342.43014 
                 −0.665269 
               
               
                 Ctnnb1 
                 PN4 
                 0.52047009 
                 0.86135196 
                 −0.1090316 
                 0.16933148 
                 178.576245 
                 −0.6438943 
               
               
                 Ash1l 
                 Astro3 
                 0.5217733 
                 0.86135196 
                 0.14203405 
                 0.2214452 
                 289.294572 
                 0.64139592 
               
               
                 Ctnnb1 
                 PN6 
                 0.52331597 
                 0.86135196 
                 0.09827286 
                 0.15353725 
                 123.928037 
                 0.64005877 
               
               
                 Setd5 
                 PN2 
                 0.52339192 
                 0.86135196 
                 −0.1524579 
                 0.23865946 
                 328.59415 
                 −0.6388094 
               
               
                 Med13l 
                 Mg1 
                 0.5262796 
                 0.86135196 
                 0.15777773 
                 0.24846606 
                 169.579268 
                 0.63500716 
               
               
                 Chd2 
                 Astro3 
                 0.52754343 
                 0.86135196 
                 −0.1913779 
                 0.30277335 
                 685.018255 
                 −0.6320832 
               
               
                 Myst4 
                 Astro1 
                 0.53112832 
                 0.86135196 
                 0.14132449 
                 0.22535003 
                 257.161689 
                 0.62713319 
               
               
                 Adnp 
                 PN4 
                 0.53196255 
                 0.86135196 
                 −0.1160729 
                 0.18537929 
                 193.584716 
                 −0.6261372 
               
               
                 Dyrk1a 
                 Astro3 
                 0.53240352 
                 0.86135196 
                 0.16104843 
                 0.25761732 
                 269.86513 
                 0.62514595 
               
               
                 Wac 
                 Mg1 
                 0.53783051 
                 0.86135196 
                 0.14401672 
                 0.23322791 
                 152.450343 
                 0.61749351 
               
               
                 Larp4b 
                 Mg2 
                 0.54422035 
                 0.86135196 
                 −0.1985139 
                 0.32711131 
                 486.914242 
                 −0.6068695 
               
               
                 Dyrk1a 
                 Astro2 
                 0.54586796 
                 0.86135196 
                 −0.1703421 
                 0.2816204 
                 229.085288 
                 −0.6048641 
               
               
                 Satb2 
                 PN5 
                 0.54686475 
                 0.86135196 
                 0.18859822 
                 0.31280172 
                 439.56242 
                 0.60293219 
               
               
                 Setd2 
                 PN4 
                 0.54791453 
                 0.86135196 
                 −0.1128338 
                 0.18752318 
                 250.238283 
                 −0.6017061 
               
               
                 Qrich1 
                 Astro2 
                 0.54987616 
                 0.86135196 
                 −0.1421928 
                 0.23746419 
                 238.726992 
                 −0.5987968 
               
               
                 Scn2a1 
                 Astro3 
                 0.55026884 
                 0.86135196 
                 −0.1345884 
                 0.22503841 
                 285.244517 
                 −0.5980684 
               
               
                 Syngap1 
                 PN6 
                 0.55127237 
                 0.86135196 
                 0.10904432 
                 0.18263618 
                 168.073906 
                 0.59705761 
               
               
                 Dyrk1a 
                 IN2 
                 0.55262344 
                 0.86135196 
                 0.10341475 
                 0.17347381 
                 87.3332077 
                 0.59614042 
               
               
                 Setd2 
                 Astro1 
                 0.55449914 
                 0.86135196 
                 0.12735678 
                 0.21522526 
                 282.926418 
                 0.59173715 
               
               
                 Dyrk1a 
                 IN1 
                 0.55478947 
                 0.86135196 
                 0.09855809 
                 0.16659089 
                 195.723534 
                 0.59161749 
               
               
                 Larp4b 
                 PN2 
                 0.55584971 
                 0.86135196 
                 −0.1742242 
                 0.29564826 
                 713.162981 
                 −0.5892955 
               
               
                 Satb2 
                 Astro2 
                 0.55965421 
                 0.86135196 
                 −0.1845858 
                 0.31605653 
                 290.947684 
                 −0.5840277 
               
               
                 Cul3 
                 Astro3 
                 0.55985119 
                 0.86135196 
                 0.13019227 
                 0.22304442 
                 304.036422 
                 0.58370557 
               
               
                 Tcf7l2 
                 Mg1 
                 0.56051561 
                 0.86135196 
                 −0.1655581 
                 0.28405635 
                 256.720203 
                 −0.5828355 
               
               
                 Wac 
                 PN6 
                 0.56178224 
                 0.86135196 
                 0.10117535 
                 0.1740238 
                 162.970282 
                 0.581388 
               
               
                 Satb2 
                 PN4 
                 0.56563382 
                 0.86135196 
                 −0.1364787 
                 0.23722424 
                 232.832508 
                 −0.575315 
               
               
                 Qrich1 
                 Mg2 
                 0.56733987 
                 0.86135196 
                 0.13881622 
                 0.24227899 
                 192.620497 
                 0.57296022 
               
               
                 Larp4b 
                 Mg1 
                 0.56809241 
                 0.86135196 
                 0.18608693 
                 0.3257364 
                 453.398346 
                 0.57128075 
               
               
                 Gatad2b 
                 Mg2 
                 0.56837789 
                 0.86135196 
                 0.17033486 
                 0.29828393 
                 311.509219 
                 0.5710494 
               
               
                 Tcf7l2 
                 PN3 
                 0.56951918 
                 0.86135196 
                 −0.142731 
                 0.25075881 
                 431.192064 
                 −0.5691965 
               
               
                 Ctnnb1 
                 Mg2 
                 0.56954701 
                 0.86135196 
                 0.13250051 
                 0.23257154 
                 188.330581 
                 0.56971935 
               
               
                 Ctnnb1 
                 Astro1 
                 0.57455161 
                 0.86512256 
                 0.11359232 
                 0.20205047 
                 220.784346 
                 0.56219777 
               
               
                 Pten 
                 Mg1 
                 0.5768521 
                 0.86512256 
                 0.1629593 
                 0.29172147 
                 293.465331 
                 0.55861262 
               
               
                 Cul3 
                 PN5 
                 0.57733689 
                 0.86512256 
                 0.14607118 
                 0.26186765 
                 347.083799 
                 0.55780536 
               
               
                 Satb2 
                 Mg1 
                 0.58222715 
                 0.86800741 
                 −0.1624275 
                 0.29481467 
                 220.577282 
                 −0.5509477 
               
               
                 Myst4 
                 PN6 
                 0.58280498 
                 0.86800741 
                 0.09359714 
                 0.17010295 
                 189.214163 
                 0.5502382 
               
               
                 Pogz 
                 Mg2 
                 0.58641234 
                 0.87059053 
                 0.16237169 
                 0.29811671 
                 286.508656 
                 0.54465812 
               
               
                 Chd8 
                 PN4 
                 0.58809279 
                 0.87059053 
                 −0.1084953 
                 0.19999306 
                 196.281911 
                 −0.5424953 
               
               
                 Med13l 
                 PN2 
                 0.59108615 
                 0.87238618 
                 −0.1432354 
                 0.26634546 
                 334.42624 
                 −0.5377807 
               
               
                 Tnrc6b 
                 PN2 
                 0.59464866 
                 0.87500854 
                 −0.1082475 
                 0.20319985 
                 283.33694 
                 −0.5327147 
               
               
                 Upf3b 
                 PN2 
                 0.60065176 
                 0.87725408 
                 −0.119003 
                 0.22712249 
                 336.350734 
                 −0.5239595 
               
               
                 Tnrc6b 
                 Mg1 
                 0.60172254 
                 0.87725408 
                 0.11253806 
                 0.21517994 
                 155.399848 
                 0.52299513 
               
               
                 Adnp 
                 Astro1 
                 0.60239263 
                 0.87725408 
                 0.1193529 
                 0.22887166 
                 318.027345 
                 0.52148396 
               
               
                 Dscam 
                 PN2 
                 0.60333597 
                 0.87725408 
                 −0.1333459 
                 0.25636602 
                 311.905546 
                 −0.5201386 
               
               
                 Kdm5b 
                 Astro1 
                 0.60600222 
                 0.87763361 
                 0.11295833 
                 0.21873998 
                 265.164233 
                 0.51640462 
               
               
                 Adnp 
                 PN2 
                 0.60870536 
                 0.87763361 
                 −0.1179118 
                 0.23008506 
                 294.925026 
                 −0.5124705 
               
               
                 Dyrk1a 
                 PN6 
                 0.60897026 
                 0.87763361 
                 −0.0937199 
                 0.18271261 
                 116.126084 
                 −0.5129363 
               
               
                 Asxl3 
                 PN6 
                 0.61880858 
                 0.88919707 
                 0.08154144 
                 0.16351083 
                 134.967287 
                 0.49869141 
               
               
                 Med13l 
                 IN2 
                 0.62153986 
                 0.89051033 
                 0.08171023 
                 0.16526207 
                 202.447606 
                 0.49442824 
               
               
                 Tnrc6b 
                 PN5 
                 0.6265434 
                 0.895062 
                 0.11204978 
                 0.23004831 
                 318.408176 
                 0.48707065 
               
               
                 Cul3 
                 PN6 
                 0.63123618 
                 0.89700953 
                 0.08088299 
                 0.16801111 
                 103.705776 
                 0.48141453 
               
               
                 Mll1 
                 Astro3 
                 0.63242543 
                 0.89700953 
                 −0.1104003 
                 0.23055997 
                 283.022482 
                 −0.4788354 
               
               
                 Asxl3 
                 Astro1 
                 0.6344336 
                 0.89700953 
                 0.09974654 
                 0.20947918 
                 218.220173 
                 0.47616445 
               
               
                 Mll1 
                 Mg2 
                 0.63646092 
                 0.89700953 
                 0.11996149 
                 0.25349095 
                 247.358705 
                 0.47323774 
               
               
                 Setd2 
                 PN6 
                 0.63976352 
                 0.89700953 
                 0.08208423 
                 0.17510165 
                 190.161713 
                 0.46878046 
               
               
                 Scn2a1 
                 Mg2 
                 0.64115736 
                 0.89700953 
                 −0.1169595 
                 0.25061287 
                 231.711082 
                 −0.4666939 
               
               
                 Dyrk1a 
                 Mg1 
                 0.64251645 
                 0.89700953 
                 0.12494974 
                 0.26876967 
                 198.337239 
                 0.46489525 
               
               
                 Med13l 
                 Astro1 
                 0.64450909 
                 0.89700953 
                 −0.1079165 
                 0.23361691 
                 262.137096 
                 −0.4619378 
               
               
                 Mbd5 
                 PN5 
                 0.64504467 
                 0.89700953 
                 0.11567117 
                 0.25088426 
                 351.991713 
                 0.46105392 
               
               
                 Pogz 
                 PN6 
                 0.64621299 
                 0.89700953 
                 −0.0907106 
                 0.19722163 
                 152.42442 
                 −0.4599424 
               
               
                 Ddx3x 
                 PN5 
                 0.65236322 
                 0.90298864 
                 0.11957824 
                 0.26524576 
                 399.901176 
                 0.45082056 
               
               
                 Ctnnb1 
                 Mg1 
                 0.65623095 
                 0.90578356 
                 0.0985363 
                 0.22089306 
                 139.310648 
                 0.44608147 
               
               
                 Med13l 
                 PN6 
                 0.66133229 
                 0.90640058 
                 0.0867966 
                 0.19763163 
                 117.896993 
                 0.43918373 
               
               
                 Chd2 
                 IN1 
                 0.66800797 
                 0.90640058 
                 0.09166642 
                 0.21363732 
                 663.530068 
                 0.42907493 
               
               
                 Ctnnb1 
                 PN5 
                 0.66818777 
                 0.90640058 
                 0.10429322 
                 0.24307399 
                 300.053246 
                 0.42905956 
               
               
                 Ddx3x 
                 Astro3 
                 0.66858888 
                 0.90640058 
                 0.10348167 
                 0.24147709 
                 281.190545 
                 0.42853619 
               
               
                 Mbd5 
                 Astro2 
                 0.66971521 
                 0.90640058 
                 −0.102649 
                 0.24034109 
                 225.978795 
                 −0.4270972 
               
               
                 Dscam 
                 Astro3 
                 0.67006192 
                 0.90640058 
                 0.10305578 
                 0.24159754 
                 253.592027 
                 0.42655972 
               
               
                 Qrich1 
                 PN5 
                 0.67177162 
                 0.90640058 
                 −0.1111911 
                 0.26220553 
                 364.547589 
                 −0.4240608 
               
               
                 Ddx3x 
                 Astro2 
                 0.67280434 
                 0.90640058 
                 −0.1116097 
                 0.26396244 
                 238.547527 
                 −0.4228243 
               
               
                 Dscam 
                 Astro2 
                 0.67659867 
                 0.90640058 
                 −0.1103218 
                 0.26413016 
                 214.684019 
                 −0.4176798 
               
               
                 Kdm5b 
                 Astro3 
                 0.67934783 
                 0.90640058 
                 0.09406856 
                 0.22734783 
                 294.082361 
                 0.41376496 
               
               
                 Kdm5b 
                 PN5 
                 0.6815008 
                 0.90640058 
                 0.10547744 
                 0.25680384 
                 380.140945 
                 0.41073156 
               
               
                 Myst4 
                 PN1 
                 0.68354758 
                 0.90640058 
                 −0.0675741 
                 0.16555972 
                 224.952228 
                 −0.4081557 
               
               
                 Pogz 
                 IN2 
                 0.68372568 
                 0.90640058 
                 0.07536628 
                 0.18484172 
                 339.730585 
                 0.40773415 
               
               
                 Fbxo11 
                 Mg1 
                 0.6839919 
                 0.90640058 
                 0.09129236 
                 0.22386815 
                 153.59168 
                 0.40779521 
               
               
                 Fbxo11 
                 Astro3 
                 0.68671096 
                 0.90640058 
                 0.08629283 
                 0.21372663 
                 272.358662 
                 0.4037533 
               
               
                 Tnrc6b 
                 Mg2 
                 0.68779724 
                 0.90640058 
                 −0.0906462 
                 0.22525947 
                 207.88535 
                 −0.402408 
               
               
                 Tcf20 
                 Astro2 
                 0.68959274 
                 0.90640058 
                 −0.0990169 
                 0.24762088 
                 249.048775 
                 −0.3998731 
               
               
                 Mbd5 
                 PN6 
                 0.69098843 
                 0.90640058 
                 −0.0659083 
                 0.16544303 
                 133.966542 
                 −0.3983744 
               
               
                 Cul3 
                 Mg2 
                 0.69182412 
                 0.90640058 
                 0.0950299 
                 0.23939421 
                 198.211022 
                 0.3969599 
               
               
                 Ank2 
                 PN4 
                 0.69734773 
                 0.91120103 
                 −0.0878675 
                 0.22561975 
                 205.179341 
                 −0.3894493 
               
               
                 Dscam 
                 PN4 
                 0.70903855 
                 0.9167956 
                 −0.0776347 
                 0.20776957 
                 208.593979 
                 −0.3736576 
               
               
                 Setd5 
                 Astro2 
                 0.71013169 
                 0.9167956 
                 0.09084722 
                 0.24415238 
                 256.216899 
                 0.37209231 
               
               
                 Stard9 
                 Mg1 
                 0.71209934 
                 0.9167956 
                 0.08370061 
                 0.226341 
                 138.003543 
                 0.36979872 
               
               
                 Ash1l 
                 Astro1 
                 0.71328325 
                 0.9167956 
                 0.07833232 
                 0.21294633 
                 260.945394 
                 0.36785004 
               
               
                 Syngap1 
                 PN2 
                 0.71548619 
                 0.9167956 
                 −0.0879997 
                 0.24122314 
                 334.934804 
                 −0.3648064 
               
               
                 Syngap1 
                 Astro2 
                 0.7172316 
                 0.9167956 
                 −0.0940068 
                 0.25927755 
                 249.375994 
                 −0.3625722 
               
               
                 Fbxo11 
                 PN2 
                 0.71756593 
                 0.9167956 
                 −0.0796295 
                 0.21994111 
                 305.007541 
                 −0.3620493 
               
               
                 Ddx3x 
                 IN2 
                 0.71941685 
                 0.9167956 
                 0.06568625 
                 0.182721 
                 404.077655 
                 0.35948933 
               
               
                 Tcf20 
                 PN5 
                 0.7200799 
                 0.9167956 
                 0.0924377 
                 0.2577549 
                 368.696866 
                 0.35862634 
               
               
                 Pogz 
                 PN2 
                 0.72119512 
                 0.9167956 
                 0.0942598 
                 0.26389691 
                 312.385529 
                 0.35718416 
               
               
                 Dscam 
                 Astro1 
                 0.72351893 
                 0.9167956 
                 0.08191233 
                 0.23126227 
                 227.946785 
                 0.35419666 
               
               
                 Setd2 
                 Astro2 
                 0.72513759 
                 0.9167956 
                 −0.0858991 
                 0.24404647 
                 261.827255 
                 −0.3519784 
               
               
                 Pogz 
                 Mg1 
                 0.7274751 
                 0.9167956 
                 −0.1010476 
                 0.28959765 
                 223.229836 
                 −0.3489242 
               
               
                 Pten 
                 PN4 
                 0.73349971 
                 0.9167956 
                 −0.0695849 
                 0.2040802 
                 192.15918 
                 −0.3409686 
               
               
                 Arid1b 
                 Mg1 
                 0.73758274 
                 0.9167956 
                 0.09462441 
                 0.28218697 
                 346.414793 
                 0.33532522 
               
               
                 Upf3b 
                 Mg2 
                 0.73872172 
                 0.9167956 
                 −0.0787076 
                 0.23563277 
                 193.231544 
                 −0.3340266 
               
               
                 Chd8 
                 Mg1 
                 0.73880366 
                 0.9167956 
                 0.08996248 
                 0.26940114 
                 186.541823 
                 0.33393506 
               
               
                 Dyrk1a 
                 PN5 
                 0.74045839 
                 0.9167956 
                 −0.093554 
                 0.28220407 
                 346.217573 
                 −0.331512 
               
               
                 Dscam 
                 Mg2 
                 0.74374681 
                 0.9167956 
                 0.08272345 
                 0.25273207 
                 214.973777 
                 0.32731678 
               
               
                 Pten 
                 Astro2 
                 0.7455054 
                 0.9167956 
                 −0.0918636 
                 0.28277339 
                 304.232644 
                 −0.3248665 
               
               
                 Chd2 
                 Astro2 
                 0.74629465 
                 0.9167956 
                 −0.1070252 
                 0.33065137 
                 591.870171 
                 −0.32368 
               
               
                 Tcf20 
                 Mg1 
                 0.7463485 
                 0.9167956 
                 0.07778108 
                 0.24006884 
                 166.511803 
                 0.3239949 
               
               
                 Asxl3 
                 Mg1 
                 0.7469197 
                 0.9167956 
                 0.07281877 
                 0.2252342 
                 148.931113 
                 0.32330246 
               
               
                 Pten 
                 Astro3 
                 0.74731023 
                 0.9167956 
                 0.08343418 
                 0.25876148 
                 357.843947 
                 0.32243664 
               
               
                 Ash1l 
                 Astro2 
                 0.74892947 
                 0.9167956 
                 0.07755759 
                 0.24205656 
                 245.383042 
                 0.320411 
               
               
                 Pten 
                 Mg2 
                 0.75275795 
                 0.9167956 
                 0.09378185 
                 0.29747301 
                 337.236655 
                 0.31526171 
               
               
                 Gatad2b 
                 PN4 
                 0.7548976 
                 0.9167956 
                 −0.0693027 
                 0.22182515 
                 368.763737 
                 −0.3124204 
               
               
                 Myst4 
                 PN5 
                 0.75635715 
                 0.9167956 
                 0.07666198 
                 0.24689496 
                 359.868385 
                 0.31050442 
               
               
                 Cul3 
                 PN4 
                 0.75735 
                 0.9167956 
                 −0.0577253 
                 0.18654884 
                 178.049098 
                 −0.3094383 
               
               
                 Wac 
                 PN4 
                 0.75775963 
                 0.9167956 
                 −0.0580044 
                 0.1878465 
                 233.23846 
                 −0.3087864 
               
               
                 Satb2 
                 Astro1 
                 0.76275127 
                 0.92056187 
                 0.08453173 
                 0.27979041 
                 322.676134 
                 0.3021252 
               
               
                 Dyrk1a 
                 Mg2 
                 0.76773544 
                 0.9237563 
                 −0.082192 
                 0.27800914 
                 261.812296 
                 −0.2956451 
               
               
                 Tnrc6b 
                 Astro3 
                 0.76936486 
                 0.9237563 
                 −0.0600881 
                 0.20472193 
                 260.9451 
                 −0.293511 
               
               
                 Asxl3 
                 IN2 
                 0.77105372 
                 0.9237563 
                 0.04328453 
                 0.14843471 
                 129.602102 
                 0.2916065 
               
               
                 Satb2 
                 Mg2 
                 0.77491926 
                 0.92422814 
                 −0.0869077 
                 0.30364009 
                 283.412765 
                 −0.2862193 
               
               
                 Ash1l 
                 Mg2 
                 0.77521993 
                 0.92422814 
                 0.06850153 
                 0.23958931 
                 215.524747 
                 0.28591227 
               
               
                 Spen 
                 PN2 
                 0.78005988 
                 0.92774112 
                 0.07033394 
                 0.25167145 
                 332.734371 
                 0.27946728 
               
               
                 Asxl3 
                 Astro3 
                 0.79373424 
                 0.93930409 
                 −0.0573414 
                 0.21907376 
                 249.416172 
                 −0.2617448 
               
               
                 Ctnnb1 
                 Astro3 
                 0.79481235 
                 0.93930409 
                 0.05501071 
                 0.21129706 
                 247.11846 
                 0.26034774 
               
               
                 Kdm5b 
                 Astro2 
                 0.79579423 
                 0.93930409 
                 −0.06438 
                 0.24850342 
                 249.518977 
                 −0.2590708 
               
               
                 Syngap1 
                 Mg2 
                 0.79745 
                 0.93930409 
                 0.06640512 
                 0.25839134 
                 197.979635 
                 0.25699438 
               
               
                 Dyrk1a 
                 Astro1 
                 0.80100366 
                 0.94033681 
                 0.06230379 
                 0.24690301 
                 229.077577 
                 0.25234114 
               
               
                 Cul3 
                 Mg1 
                 0.80216487 
                 0.94033681 
                 0.05717643 
                 0.22777634 
                 140.678597 
                 0.25102006 
               
               
                 Wac 
                 Mg2 
                 0.80926443 
                 0.94435396 
                 0.05900248 
                 0.24413841 
                 212.124696 
                 0.24167635 
               
               
                 Wac 
                 Astro1 
                 0.8138117 
                 0.94435396 
                 0.0490153 
                 0.20788182 
                 227.884321 
                 0.23578445 
               
               
                 Fbxo11 
                 Astro2 
                 0.81490116 
                 0.94435396 
                 −0.054759 
                 0.23363769 
                 230.864646 
                 −0.2343756 
               
               
                 Tcf20 
                 Astro3 
                 0.81502562 
                 0.94435396 
                 −0.0530461 
                 0.22653983 
                 293.399521 
                 −0.234158 
               
               
                 Arid1b 
                 Astro2 
                 0.81583699 
                 0.94435396 
                 0.06759268 
                 0.29006806 
                 508.015379 
                 0.23302353 
               
               
                 Upf3b 
                 PN5 
                 0.81799039 
                 0.94435396 
                 0.05782534 
                 0.25109455 
                 372.627703 
                 0.23029311 
               
               
                 Kdm5b 
                 Mg1 
                 0.81908252 
                 0.94435396 
                 −0.0568919 
                 0.24838644 
                 187.799217 
                 −0.2290459 
               
               
                 Setd5 
                 PN5 
                 0.82287605 
                 0.94650062 
                 −0.0592266 
                 0.26439819 
                 372.088306 
                 −0.2240054 
               
               
                 Fbxo11 
                 PN4 
                 0.83293853 
                 0.95439023 
                 −0.037647 
                 0.1782601 
                 214.55213 
                 −0.2111912 
               
               
                 Gatad2b 
                 PN5 
                 0.8342434 
                 0.95439023 
                 0.05970686 
                 0.285145 
                 424.120169 
                 0.20939121 
               
               
                 Tnrc6b 
                 Astro2 
                 0.83811874 
                 0.95439023 
                 −0.0457778 
                 0.2238068 
                 220.938769 
                 −0.2045414 
               
               
                 Pten 
                 Astro1 
                 0.83839476 
                 0.95439023 
                 0.05116742 
                 0.25069269 
                 338.496032 
                 0.20410417 
               
               
                 Mll1 
                 PN5 
                 0.84016055 
                 0.95439023 
                 0.05065626 
                 0.25097432 
                 349.219732 
                 0.20183844 
               
               
                 Satb2 
                 PN2 
                 0.84142159 
                 0.95439023 
                 −0.0578021 
                 0.28870393 
                 379.751005 
                 −0.2002125 
               
               
                 Tnrc6b 
                 Astro1 
                 0.85088185 
                 0.9628917 
                 0.03692198 
                 0.19618528 
                 236.127794 
                 0.18819956 
               
               
                 Myst4 
                 Astro2 
                 0.86832382 
                 0.97488347 
                 −0.0425353 
                 0.25629171 
                 241.569514 
                 −0.1659644 
               
               
                 Mbd5 
                 Astro3 
                 0.86874708 
                 0.97488347 
                 −0.0363657 
                 0.21985265 
                 266.629192 
                 −0.1654094 
               
               
                 Arid1b 
                 PN2 
                 0.87010778 
                 0.97488347 
                 −0.0454905 
                 0.27803367 
                 449.574964 
                 −0.163615 
               
               
                 Chd8 
                 PN2 
                 0.87215093 
                 0.97488347 
                 0.03987962 
                 0.24760521 
                 307.360431 
                 0.1610613 
               
               
                 Tcf7l2 
                 Astro3 
                 0.87223368 
                 0.97488347 
                 0.04157664 
                 0.25835202 
                 381.015737 
                 0.16093018 
               
               
                 Wac 
                 PN5 
                 0.87525192 
                 0.97488347 
                 0.04007667 
                 0.25510151 
                 366.220707 
                 0.15710088 
               
               
                 Ash1l 
                 PN4 
                 0.87540557 
                 0.97488347 
                 −0.0279633 
                 0.17812059 
                 206.349643 
                 −0.1569908 
               
               
                 Setd2 
                 PN5 
                 0.88565354 
                 0.97691898 
                 0.03652111 
                 0.25378204 
                 362.715728 
                 0.14390736 
               
               
                 Myst4 
                 Mg2 
                 0.88831642 
                 0.97691898 
                 0.03565465 
                 0.25362142 
                 243.19471 
                 0.14058215 
               
               
                 Tcf20 
                 Astro1 
                 0.89292861 
                 0.97691898 
                 0.02935694 
                 0.21789295 
                 260.417272 
                 0.13473103 
               
               
                 Dscam 
                 PN6 
                 0.89594773 
                 0.97691898 
                 0.02494567 
                 0.19038756 
                 136.592904 
                 0.13102574 
               
               
                 Ank2 
                 PN5 
                 0.89801554 
                 0.97691898 
                 0.04006933 
                 0.3124074 
                 357.055839 
                 0.12825985 
               
               
                 Adnp 
                 Astro3 
                 0.89866209 
                 0.97691898 
                 0.03016065 
                 0.23665236 
                 339.176039 
                 0.12744707 
               
               
                 Tnrc6b 
                 PN6 
                 0.90146418 
                 0.97691898 
                 0.01861151 
                 0.15005507 
                 143.46784 
                 0.12403119 
               
               
                 Adnp 
                 Mg1 
                 0.90243232 
                 0.97691898 
                 −0.0296441 
                 0.24148852 
                 186.845529 
                 −0.1227559 
               
               
                 Upf3b 
                 PN4 
                 0.90251466 
                 0.97691898 
                 −0.0227481 
                 0.18551679 
                 230.999492 
                 −0.12262 
               
               
                 Med13l 
                 Astro2 
                 0.90329684 
                 0.97691898 
                 −0.0322817 
                 0.26542972 
                 250.873452 
                 −0.1216206 
               
               
                 Myst4 
                 PN4 
                 0.90451212 
                 0.97691898 
                 −0.0218829 
                 0.18222761 
                 249.200966 
                 −0.1200856 
               
               
                 Setd5 
                 Mg1 
                 0.90597949 
                 0.97691898 
                 −0.0283612 
                 0.23979317 
                 184.750567 
                 −0.1182734 
               
               
                 Stard9 
                 Astro2 
                 0.90681803 
                 0.97691898 
                 −0.027422 
                 0.23399677 
                 217.539235 
                 −0.1171895 
               
               
                 Tcf20 
                 Mg2 
                 0.9069012 
                 0.97691898 
                 −0.029289 
                 0.250169 
                 230.547513 
                 −0.1170767 
               
               
                 Asxl3 
                 Astro2 
                 0.90762753 
                 0.97691898 
                 −0.0278242 
                 0.23951093 
                 211.302912 
                 −0.1161708 
               
               
                 Spen 
                 Astro3 
                 0.91209925 
                 0.97691898 
                 0.02609224 
                 0.23614125 
                 270.913982 
                 0.11049421 
               
               
                 Ddx3x 
                 PN2 
                 0.91216931 
                 0.97691898 
                 0.026839 
                 0.24316283 
                 388.453514 
                 0.11037458 
               
               
                 Asxl3 
                 Mg2 
                 0.91312019 
                 0.97691898 
                 −0.0257891 
                 0.23608315 
                 206.896159 
                 −0.1092373 
               
               
                 Tcf20 
                 PN4 
                 0.91616662 
                 0.9780428 
                 −0.0198659 
                 0.1885025 
                 213.790192 
                 −0.1053882 
               
               
                 Tcf7l2 
                 PN1 
                 0.91906827 
                 0.97900751 
                 0.02647334 
                 0.26039243 
                 430.460985 
                 0.10166708 
               
               
                 Setd5 
                 Mg2 
                 0.92324484 
                 0.97981268 
                 0.02401188 
                 0.24896202 
                 241.33821 
                 0.09644797 
               
               
                 Syngap1 
                 Astro1 
                 0.92382339 
                 0.97981268 
                 −0.0218443 
                 0.22823569 
                 266.088466 
                 −0.0957094 
               
               
                 Scn2a1 
                 PN6 
                 0.92854312 
                 0.98151797 
                 0.01553852 
                 0.17297604 
                 148.338353 
                 0.08983045 
               
               
                 Ddx3x 
                 PN4 
                 0.92943743 
                 0.98151797 
                 0.01788263 
                 0.20177379 
                 299.739093 
                 0.08862713 
               
               
                 Mbd5 
                 Mg2 
                 0.93457445 
                 0.98482039 
                 0.02043943 
                 0.24869287 
                 214.06079 
                 0.08218745 
               
               
                 Tcf7l2 
                 Astro2 
                 0.93725549 
                 0.98552616 
                 0.02224033 
                 0.28230588 
                 323.973359 
                 0.07878095 
               
               
                 Setd5 
                 Astro3 
                 0.94408912 
                 0.98649408 
                 0.0156786 
                 0.22337452 
                 301.863442 
                 0.07018973 
               
               
                 Ank2 
                 Astro3 
                 0.94420207 
                 0.98649408 
                 −0.0197268 
                 0.28171107 
                 497.705054 
                 −0.0700248 
               
               
                 Larp4b 
                 PN5 
                 0.94421577 
                 0.98649408 
                 0.02136092 
                 0.30514278 
                 570.959429 
                 0.07000302 
               
               
                 Tcf7l2 
                 Astro1 
                 0.94811979 
                 0.98829569 
                 −0.0163352 
                 0.25087837 
                 372.234954 
                 −0.065112 
               
               
                 Mll1 
                 PN6 
                 0.95214938 
                 0.98829569 
                 0.01002708 
                 0.16678757 
                 135.720847 
                 0.06011889 
               
               
                 Setd5 
                 PN4 
                 0.95504745 
                 0.98829569 
                 0.01099067 
                 0.1947672 
                 234.675057 
                 0.05642978 
               
               
                 Tcf7l2 
                 PN5 
                 0.95601699 
                 0.98829569 
                 0.01872843 
                 0.33941191 
                 520.43967 
                 0.05517906 
               
               
                 Kdm5b 
                 PN4 
                 0.95859711 
                 0.98829569 
                 0.01001849 
                 0.19280908 
                 279.895275 
                 0.05196066 
               
               
                 Ddx3x 
                 Mg2 
                 0.96062219 
                 0.98829569 
                 0.01260716 
                 0.25502181 
                 197.183768 
                 0.04943561 
               
               
                 Spen 
                 Mg2 
                 0.96215406 
                 0.98829569 
                 0.01228317 
                 0.25857764 
                 227.964511 
                 0.04750283 
               
               
                 Arid1b 
                 PN6 
                 0.96284218 
                 0.98829569 
                 0.01041347 
                 0.22333249 
                 289.065857 
                 0.04662766 
               
               
                 Ank2 
                 Mg1 
                 0.9649606 
                 0.98829569 
                 −0.0137277 
                 0.31221077 
                 276.183992 
                 −0.0439694 
               
               
                 Myst4 
                 PN2 
                 0.96610946 
                 0.98829569 
                 −0.0094631 
                 0.22255594 
                 334.756277 
                 −0.0425201 
               
               
                 Qrich1 
                 Astro3 
                 0.97462951 
                 0.9925345 
                 −0.0069148 
                 0.21723629 
                 281.456446 
                 −0.0318308 
               
               
                 Myst4 
                 Astro3 
                 0.97585756 
                 0.9925345 
                 −0.0071017 
                 0.2344636 
                 284.865655 
                 −0.0302893 
               
               
                 Stard9 
                 Mg2 
                 0.97632985 
                 0.9925345 
                 −0.0070777 
                 0.23823155 
                 191.969124 
                 −0.0297092 
               
               
                 Pogz 
                 PN5 
                 0.98551667 
                 0.99755377 
                 −0.0053377 
                 0.29383165 
                 361.796228 
                 −0.0181657 
               
               
                 Med13l 
                 PN5 
                 0.98599396 
                 0.99755377 
                 0.00515119 
                 0.2932471 
                 393.983741 
                 0.01756602 
               
               
                 Arid1b 
                 Astro1 
                 0.98737465 
                 0.99755377 
                 −0.0041315 
                 0.26097358 
                 588.124274 
                 −0.0158309 
               
               
                 Spen 
                 PN4 
                 0.98983057 
                 0.99791738 
                 −0.0026202 
                 0.2053524 
                 229.506518 
                 −0.0127597 
               
               
                 Stard9 
                 PN5 
                 0.99180769 
                 0.99791738 
                 0.00253996 
                 0.24718726 
                 333.270175 
                 0.01027543 
               
               
                 Qrich1 
                 PN4 
                 0.99425614 
                 0.99833096 
                 0.0013827 
                 0.19185089 
                 219.186257 
                 0.00720714 
               
               
                 Stard9 
                 Astro3 
                 0.99788991 
                 0.99956811 
                 0.0005666 
                 0.21403846 
                 256.769662 
                 0.00264718 
               
               
                 Asxl3 
                 PN2 
                 0.99956811 
                 0.99956811 
                 −0.0001197 
                 0.22086453 
                 299.528783 
                 −0.0005417 
               
               
                   
               
               
                 *Used FindVariableGenes with x.low.cutoff = 1, x.high.cutoff = 5 on the combined dataset 
               
               
                 **Used FindVariableGenes separately on each batch (with x.low.cutoff = 1, x.high.cutoff = 5), then only kept those that occurred in al least a certain number of batches (specified in column E). So. if this column = 4, means that variable genes have to be variable in at least 4 batches. 
               
               
                 ***For those datasets where calculated variable genes on each batch were calculated and combined, this column has the number of batches required to have a genes as variable for it to be kept 
               
            
           
         
       
     
     Perturbations in 9 ASD/ND genes (Adnp, Ank2, Ash11, Chd8, Gatad2b, Pogz, Scn2a1, Stard9, and Upf3b) had significant effects across 5 modules ( FIG. 2B , highlighted circles, compared to the GFP control, FDR corrected P&lt;0.05): a module associated with projection neurons of Layer 4 and 5 (PN1, affected by perturbations in Adnp, Ash11, Scn2a1, and Stard9); modules representing two distinct homeostatic signatures in astrocytes (Astrol affected by perturbation of Scn2a1, and Astro3 affected by perturbations of Chd8, Pogz, and Upf3b); a module associated with oligodendrocyte progenitor cells (ODC1, Chd8 and Gatad2b); and a module associated with Ndnf+interneurons Ank2) ( FIGS. 2C and 10A-10F ). 
     Notably, the oligodendrocyte progenitor module (ODC1) also had a significant amount of its variation across the oligodendrocyte cell cluster explained by the perturbation state overall (van der Waerden test, a non-parametric alternative to ANOVA analysis, FDR corrected P&lt;0.05) ( FIG. 9C ), suggesting that this module represents convergent effects across different perturbed genes. Collectively, the data indicate that a selected group of perturbations was able to affect recurrent gene modules with cell-type specificity and point to some convergent effects across diverse ASD/ND risk genes. 
     Example 5—Single Perturbation of Ank2 Confirms Perturb-Seq Effect on an Interneuron Gene Expression Module 
     In the multiplex in vivo Perturb-Seq results, Ank2 perturbation led to increased expression of an interneuron module (IN1) (FDR corrected P&lt;0.05,  FIGS. 14A-14E ). This module was strongly correlated with a subcluster of inhibitory interneurons expressing Ndnf ( FIGS. 14C-14D ) and contains genes such as Kcnq5 (a voltage-gated potassium channel) and Gabbr2 (GABA receptor subunit) ( FIG. 10B  and Table 4). To validate the finding from the pooled, Perturb-Seq experiment, performed a single perturbation targeting either Ank2 or GFP (control) was performed, followed by scRNA-seq of neocortical cells at P7, resulting in 2,943 and 1,716 high-quality cells, respectively. 
     The individual perturbation experiment confirmed the results from the pooled Perturb-Seq screen. Ank2-perturbed cells were present across all cell types and overall proportions of cells were not significantly changed ( FIG. 14B ). Within the Ndnf+interneurons, Ank2 perturbation led to upregulation of the IN1 module (FDR corrected P&lt;0.05,  FIG. 14E ), confirming the Perturb-Seq result. This finding indicates that multiplexing perturbations in the pooled approach does not significantly distort the results observed for an individually perturbed gene. 
     Ank2 encodes an ankyrin protein and is expressed broadly in excitatory and inhibitory neurons as well as glial cells in the brain (22). Studies examining Ank2 loss-of-function suggest that it is involved in axonal morphology, connectivity, and calcium signaling in excitatory neurons (23-26). This Perturb-Seq data suggests a role of Ank2 in the Ndnf+interneuron subtype during cortical development, in addition to its known roles in excitatory neurons. 
     Example 6—the ASD/ND Risk Genes Chd8 and Gatad2b Alter Gene Modules in Oligodendrocyte Progenitors 
     In the Perturb-Seq experiment, Chd8 and Gatad2b perturbations significantly decreased the expression of the ODC1 module in the oligodendrocyte cluster ( FIGS. 3A-3D , FDR corrected P&lt;0.05; see alternative measurement of effect size  FIG. 15A , estimated by log transcripts per million (TPM) gene expression difference). The ODC1 module is highly expressed in cycling cells and oligodendrocyte precursor cells (OPC), and lowly expressed in committed oligodendrocyte progenitor cells (COP) and newly formed oligodendrocytes (NFOL), suggesting that this module is linked to oligodendrocyte maturation ( FIG. 3A ), and therefore that perturbation in Chd8 and Gatad2b might accelerate oligodendrocyte maturation. This is consistent with recent reports that Chd8 loss-of-function potentiates an impaired OPC development phenotype caused by deletion of Chd7 (27). 
     This result was further investigated and validated by examining oligodendrocyte development in a Chd8 germline heterozygous mutant model (as homozygous mutation is embryonic lethal (28)), using several orthogonal methods. First, in situ hybridization was used for two canonical OPC markers known to be involved in fate specification, Cspg4 (a member of the ODC1 module) and Pdgfra. Both were downregulated in P7 Chd8+/−cortex ( FIGS. 3E and 15B-15D ), consistent with the in vivo Perturb-Seq results demonstrated here and elsewhere herein. 
     Second, immunohistochemistry was used to examine a later developmental time point, P11. OPC cell number (e.g., PDGFRA+ cells) did not show significant differences between the WT and Chd8+/−littermates, also consistent with in vivo Perturb-Seq; however, cells positive for the MBP protein, a marker of myelinating oligodendrocytes, were increased in number and displayed elevated MBP levels in the Chd8+/−mutant (FDR corrected P&lt;0.05, nonparametric ANOVA test) ( FIG. 3F ). In combination with the Perturb-Seq result showing reduction in the signature of oligodendrocyte progenitors and of the progenitor-expressed ODC1 module in Chd8-perturbed cells, this suggests that Chd8 perturbation may result in acceleration of the progressive increase in MBP levels that occurs postnatally. These data further demonstrate that in vivo Perturb-Seq has the power to identify cell type-specific molecular changes similar to those observed in a single-gene, germline-modified mouse model. 
     Example 7—Perturb-Seq Gene Modules are Conserved Between Human and Mouse 
     To establish whether the perturbed gene modules identified in the mouse cerebral cortex are conserved in human cells, the expression of each module across multiple scRNA-seq datasets from human tissues was examined: adult human cortex (29), ASD donor cortex with matched controls (30), fetal human cortex (31), and 3 month and 6 month-old human brain organoids (32) ( FIGS. 4A-4B ). In the fetal brain and the 3-month brain organoid samples, glial cell types were sparsely represented due to the early developmental stages of the samples ( FIG. 16A ). Human genes that had 1:1 orthologs to the mouse genes in each module were identified, and asked whether the modules were conserved, using two metrics: whether the orthologous genes were also expressed in the corresponding cell type in the human datasets, and whether the expression of the genes in each module co-varied across single cells (as estimated by correlation), reflecting the degree of “modularity” of these mouse gene programs in humans. 
     The expression of each module was largely conserved in all human datasets, with different modules showing distinct levels of conservation of expression in each dataset ( FIG. 4A ). Some modules like PN1, PN2, and PN5 displayed high levels of conservation of expression (with at least 75% of the genes in these modules being expressed by at least 5% of cells in the corresponding associated cell type) across all datasets. The proportions of the genes expressed in the corresponding cell types in human tissues were generally lower than in mouse tissues ( FIG. 16B ). 
     It was further calculated whether the co-variation of expression of the genes in each module (their “modularity”) was also comparable in humans. To do so, for each module and each dataset the average pairwise expression correlation coefficient between the genes in a given module was calculated and compared to a module-specific null-distribution based on random gene sets with similar expression levels, to calculate both a P-value for the correlation of these modules and a normalized correlation coefficient. 8 out of 14 modules showed greater intra-module correlation than a comparable random gene set in the adult human brain dataset from Hodge et al (29) ( FIG. 4B ). Correlation also increased with the age of the human samples across brain regions of the BrainSpan dataset (9) ( FIGS. 4C-4E and 17A-17E ). As a control, the same approach was used to calculate the expression and modularity of each gene module in non-associated cell types. It was found that the modularity was decreased in non-associated cell types ( FIGS. 16D-16E ), reflected by both the proportion of comparisons with significant correlation and by the strength of the significant correlations, suggesting that these modules reflect cell type-specific effects. 
     Altogether, these results suggest that expression and modularity of most gene modules in the mouse are conserved in human brain tissue, pointing at potential shared functions and suggesting that processes identified as affected in the Perturb-Seq experiments demonstrated herein are relevant to biological processes that may be developmentally regulated in the human brain. 
     Example 8—Mouse Perturb-Seq Results are Correlated with Expression Changes in ASD Patient Brain Tissues 
     Finally, it was explored whether the effects observed in mouse Perturb-Seq may be similar to changes observed in postmortem brains of ASD patients. To this end, the data demonstrated herein was compared to a single-nucleus RNA-seq (snRNA-seq) dataset of postmortem ASD brain samples (30), and bulk RNA-seq of postmortem psychiatric disorder brain samples from the PsychEncode project (33). 
     Using a dataset of snRNA-seq profiles from 15 ASD donors and 16 controls (30), defined differentially expressed (DE) genes in each cell type were defined using a statistically conservative pseudobulk-based analysis with DESeq2 (34, 35), correcting for age, sex, and patient-to-patient variability. Genes were identified that were differentially expressed between patients and controls in at least one of three major cell types (inhibitory neurons, excitatory neurons, or oligodendrocytes) with FDR &lt;0.2, and selected those that have 1:1 orthologs in mice, resulting in 14 genes ( FIG. 4F ). 
     These 14 genes were then compared to the Perturb-Seq data and asked if these ASD-patient DE genes were also affected by the 35 ASD risk gene perturbations in the dataset. The effects of all 35 perturbations were aggregated, and it was asked whether the aggregated gene expression changes agreed more strongly with the gene expression changes in the ASD patient data than would be expected by chance. For each ASD patient DE gene, its mouse orthologue was taken and the median fold change of expression (log FC) over all perturbations in the Perturb-Seq data was calculated. This log FC was then compared with the corresponding log FC in the ASD patient data and generated an agreement score for each gene, defined as a high median log FC and a similar direction of change as in the human data. Genes were then binned by their expression and each ASD patient DE gene was compared to others in the same bin to extract p-values (with FDR correction). From this analysis, two genes were identified, SST in interneurons and NRN1 in excitatory neurons, both of which showed decreased expression in ASD patients and were likewise significantly decreased in expression across the panel of perturbations (FDR &lt;0.1), albeit with different effect sizes ( FIG. 4F ). This indicates that despite the different developmental stages, high clinical heterogeneity in ASD, and patient genetic diversity, similar genes and cell types can be identified as affected in both the analyses herein and in studies of human patient tissue. 
     The 14 gene modules reported in the PsychEncode study of 700 bulk RNA-seq samples of human cortex from a panel of psychiatric disorders was also analyzed (33). 6 of the 14 modules previously reported to be altered in the ASD patients in the PsychEncode analysis were also significantly affected across 8 of the ASD/ND risk gene perturbations ( FIG. 18 ). Although these analyses are limited by the relatively few available datasets of ASD patient brain samples, they suggest that these Perturb-Seq experiments can identify gene program abnormalities seen in human ASD patients. 
     Example 8—Discussion of Examples 1-7 
     In vivo Perturb-Seq can serve as a scalable tool for systems genetic studies of large gene panels to reveal their cell-intrinsic functions at single-cell resolution in complex tissues. In this example, at least the application of in vivo Perturb-Seq to ASD/ND risk genes in the developing brain was demonstrated. This method can be applied across diverse diseases and tissues. 
     ASD/ND affects brain function profoundly, but its cellular and molecular substrates are not yet defined. The large number of highly penetrant de novo risk genes implicated through human genetic studies offers an entry point to identify the cell types, developmental events, and mechanisms underlying ASD/ND. However, this requires scalable methods to define the function of risk-associated genes with cell-type specificity. Using Perturb-Seq to functionally test large gene sets in the developing embryo, gene expression changes were observed to be linked to ASD/ND genes in different cell types and processes. Within the power of the analysis that can be achieved with the number of cells that can be reasonably sequenced, it was found that some recurrent modules are affected across more than one ASD/ND risk gene perturbation. Without being bound by theory, it is likely that this represents an underestimation of the number of convergent modules across perturbations which might be revealed by larger-scale experiments using greater numbers of cells. 
     Ank2 encodes an ankyrin protein and is expressed broadly in excitatory and inhibitory neurons as well as glial cells in the brain (22). Ankyrin homologs interact with ion channels in many neuronal types, and Ankyrin-G has been shown to stabilize GABAergic synapses (36). The roles of Ank2 in the brain have largely been studied in the context of excitatory neurons. Ank2 loss-of-function results in hypoplasia of the corpus callosum and pyramidal tract, and ultimately optic nerve degeneration (23), suggesting that it is required in the maintenance of premyelinated axons in excitatory neurons in early neurodevelopment. Ank2 mutants showed misregulation of intracellular calcium homeostasis and calcium channel expression in excitatory neurons (24, 25), as well as increased axonal branching and ectopic connectivity (26). The Perturb-Seq data in at least examples 1-7 suggests an additional role of Ank2 in the Ndnf+interneuron subtype, along with its known roles in excitatory neurons. 
     In addition to neurons, oligodendrocytes and astrocytes were also affected by several perturbations. Oligodendrocytes modulate and consolidate neural circuit refinement, and abnormal maturation of oligodendrocytes may be linked to long-lasting changes in neural wiring and brain function (37). One of the risk genes, Chd8, encodes a protein that binds directly to β-catenin to recruit histone proteins and negatively regulates the Wnt signaling pathway, which plays a crucial role in neuronal progenitor proliferation and differentiation in the forebrain (38-41). The results in these examples at least showed that Chd8 modulates gene modules for oligodendrocyte differentiation and maturation, consistent with previously reported ChIP-Seq results showing that CHD8 interacts directly with OPC maturation genes at perinatal stages of development (27, 42). 
     Although these examples focused on the perinatal neocortex in this study, in vivo Perturb-Seq can be applied to study gene functions systematically across other tissues and developmental ages to reveal tissue-specific as well as broadly-distributed gene functions. This approach can uncover both the impact of individual disease-associated genes and of combinations of genes and the overall set of processes that they affect. These findings underscore the importance of using single-cell profiles as a rich, comprehensive, and interpretable phenotypic readout. With advances in other single-cell profiling approaches (e.g., single-cell ATAC-seq (43), single-cell multi-omics (44), and spatial genomics (45, 46)), in vivo Perturb-Seq can be coupled in the near future with diverse readouts to better define the function of disease-risk associated variants, from molecular mechanisms to non-cell autonomous effects in tissues. Spatial transcriptomics in combination with in vivo Perturb-Seq can be used to uncover non-cell autonomous effects. In vivo Perturb-Seq can allow for, inter alia, elucidation and understanding of pathways and cell types affected in heterogenous genetic pathologies, directing downstream studies, informing the development of refined models for genetic disorders, and mechanistic studies as interest moves from genetic variants to function. 
     Example 9—Methods for Examples 1-7 
     Methods Summary 
     In Vivo Perturb-Seq Experiment 
     The backbone plasmid contains antiparallel cassettes of two gRNAs (Table 6) under mouse U6 and human U6 promoters, and the EF1a promoter to express puromycin, BFP, and a polyadenylated barcode unique to each perturbation. Cloning and lentiviral packaging of the 38 vectors were done individually. 
     All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committees (IACUC) of Harvard University and of the Broad Institute of MIT and Harvard. In utero lentiviral injection into the lateral ventricles was performed at E12.5 in Cas9 transgenic mice (14) (4-6 month old, Jax #026179), and each single-cell library was made by combining the BFP +  cells from 1-3 litters (4-20 animals) of P7 animals harvested on the same day. Tissue dissociation was performed with the Papain Dissociation kit (Worthington, #LK003152). The FACS-purified cells were sorted into cold Hibernate A/B27 medium and subjected to single-cell RNA sequencing library preparation. The analysis includes 17 independent libraries of Perturb-Seq cells. 
     Single-cell RNA sequencing libraries were created using the Chromium Single Cell 3′ Solution v2 kit (10× Genomics) following the manufacturer&#39;s protocol. Each library was sequenced with Illumina NextSeq high-output 75-cycle kit with sequencing saturation above 70%. Dial-out PCR was performed to extract the perturbation barcode in each cell. 
     Perturbation barcodes were identified by two complementary methods. First, the dial-out sequences were used to create a cell-by-perturbation UMI count matrix by a modification of from the original Perturb-Seq work (12). In addition, barcode sequences were extracted from the 10× Genomics Cell Ranger bam file. Reads were then assigned to the perturbation they mapped best. Cell barcodes and UMIs were extracted, and a cell-by-perturbation UMI count matrix was created. Then, only cells for which either i) the assigned 10× and dialout perturbations agree or ii) the cell was assigned to a perturbation by one method but not assigned to any perturbation in the other were kept. 
     Perturb-Seq Analysis 
     UMI count data was loaded into R and processed using the Seurat v 2.2 package (47). Clusters were assigned to cell types based on marker genes from the literature, mousebrain.org (16), and DropViz.com (22). Only on cells of 5 key types (projection neurons, inhibitory neurons, oligodendrocytes, microglia/macrophages, and astroglia) were focused on and rest were removed. 
     WGCNA and Structural topic modelling (STM) were performed for each cell cluster based on the published pipelines (20, 21). Linear regression was used to test the relationship between perturbations and WGCNA gene scores, correcting for batch and number of genes. To test for correlations between perturbations and topics, the theta matrix (the matrix containing proportions of topics per cell) was extracted from the STM matrix. For each topic, linear regression was used to test how the per-cell proportions for each topic related to perturbations (after setting GFP to be the reference perturbation), correcting for nGene and batch. 
     RNA In Situ Hybridization and Immunohistochemistry 
     Multiplexed RNAscope fluorescent in situ hybridization and immunohistochemistry was performed on fixed-frozen tissue. Probes against the following mRNAs were used: Pdgfra, Cspg4, and Fezf2 (ACDBio). The antibodies and dilutions were: Mouse anti-NeuN antibody (mab377, 1:500; Millipore), Mouse anti-GS antibody (mab302, 1:500; Millipore), Goat anti-Pdgfra antibody (AF1062, 1:200; R&amp;D System), Rabbit Ibal antibody (019-19741, 1:400; Wako), Chicken anti-GFP antibody (ab16901, 1:500; Millipore), Mouse anti-Satb2 (ab51502, 1:50; Abcam), Rat anti-Ctip2 (ab18465, 1:100, Abcam), Rabbit anti-Sox6 (ab30455, 1:500; Abcam), Rat anti-Mbp (mab386, 1:100; Millipore). The staining, imaging, and quantifications were double-blinded. 
     Analysis of Human Single Nucleus or Single Cell RNA-Seq Data 
     For each single cell/nucleus human dataset, the UMI count matrix and metadata were downloaded and processed with Seurat to create Seurat objects. Cell types were extracted from the metadata, and combined into more general cell types, namely: Microglia, Astroglia (including Radial Glia), Inhibitory neurons, Excitatory neurons, Oligodendrocytes, and other. For differential expression analysis for data from Velmeshev et al (30), we removed data from all individuals of &lt;12 years of age and separated PFC and ACC regions. For each cell type in each region a pseudobulk profile was constructed and genes expressed in &lt;5% of cells or with &lt;10 reads were removed. DESeq2 v 1.20.0 (35) was then used to perform differential expression analysis between the ASD patients and the controls, correcting for sex and age. All genes with 1:1 mouse orthologs (BioMart) were extracted and the FDR corrected P-values were calculated on these genes for both ACC and PFC. Only analysis on the PFC yielded significant hits, which are presented in  FIG. 4F . 
     To compare these results to the Perturb-Seq data, for each human DE gene, an agreement score was calculated by taking the absolute value of its mouse orthologues&#39; median log FC over all perturbations (calculated with Limma) and giving it a positive sign if its direction agreed with that of the human data, a negative sign otherwise. Finally, genes were binned by expression, and p-values were calculated for each gene by comparing the agreement scores to other genes in the same bin. 
     Further method details are set forth below. 
     Lentiviral Vector Construction and Production 
     Lentiviral vectors were constructed as previously reported (11-13). The backbone plasmid contains antiparallel cassettes of two gRNAs (Table 6) under mouse U6 and human U6 promoters, and the EF1a promoter to express puromycin, BFP, and a polyadenylated barcode unique to each perturbation. Cloning of the 38 vectors were done individually. Association of each gRNA set and perturbation barcode was established by Sanger sequencing. The gRNA designs were defined using the online tool at benchling.com (48). Each lentivirus was packaged individually with the V2 helper plasmids (49), and the functional titer was measured individually through HEK293 cell infection and FACS measurement of the BFP+ population before pooling equally for ultracentrifugation. The functional titer of the final lentivirus was &gt;5×10 9  U/mL for in utero ventricular injection and transduction. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 gRNA design for the ASD/ND risk gene perturbations. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
                 SEQ 
                   
                 SEQ 
               
               
                   
                   
                 ID 
                   
                 ID 
                   
                 ID 
               
               
                 gene 
                 Guide1-179 
                 NO: 
                 Guide2-117 
                 NO: 
                 Perturbation barcode 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 ADNP 
                 CCTGGGCACAAATGCCCGAG 
                 1 
                 TTTGAAAAACACTACATGGG 
                 41 
                 CTAGTTACTTTAGATAGG 
                 81 
               
               
                   
               
               
                 ANK2 
                 GCATTTCTGCGACTACACTG 
                 2 
                 TGTTCCTGAGACAATGACGG 
                 42 
                 ACTAAAGCTGCATCGCGG 
                 82 
               
               
                   
               
               
                 ANKRD11 
                 CTGCACGAGGCGTGTAACCG 
                 3 
                 GCACCGAGCAGCTATCCGAG 
                 43 
                 GTCTTGTTGGAGTCGAGT 
                 83 
               
               
                   
               
               
                 ARID1B 
                 GTACCCATCCCATACAACTG 
                 4 
                 CCCATGATGAGGAGCTACGG 
                 44 
                 GTTGTCCTGTTGGTCTGG 
                 84 
               
               
                   
               
               
                 ASH1L 
                 ACTATGAGACTCACTAACTG 
                 5 
                 ACTTCTCTTGATGTGATGGG 
                 45 
                 CGGGCGAATGGGAACCTG 
                 85 
               
               
                   
               
               
                 ASXL3 
                 TCACACTAACACTCGAGTCG 
                 6 
                 AGATTGCAGCCTTACGAACA 
                 46 
                 GGTTTTGTTGGGCGACCA 
                 86 
               
               
                   
               
               
                 CHD2 
                 TCAGAAGACGAACAGGAACA 
                 7 
                 TAAGGACAAAAGCCAAGAGG 
                 47 
                 TCATTCATCCGGCCTATC 
                 87 
               
               
                   
               
               
                 CHD8 
                 TTTCAATCCAGACTACGTAG 
                 8 
                 TGCCCTATGAGGACAGTACG 
                 48 
                 GTGTTGCGCCCTCTTCAA 
                 88 
               
               
                   
               
               
                 CTNNB1 
                 CCATTCATAAAGGACTTGGG 
                 9 
                 GATTAACTATCAGGATGACG 
                 49 
                 AGAAGTGATGGTGTCAAG 
                 89 
               
               
                   
               
               
                 CUL3 
                 ATCCAGCGTAAGAATAACAG 
                 10 
                 TATGTCTCTAATCATCACCA 
                 50 
                 CATCTCCCTGATGGCGTA 
                 90 
               
               
                   
               
               
                 DDX3X 
                 ATGACAAAGACAGTTCAGGG 
                 11 
                 AAAGAGGTGGAAATAGTCGC 
                 51 
                 AAGGTACACCTGGTTTGA 
                 91 
               
               
                   
               
               
                 DSCAM 
                 AGTGACGTACGCCTCCACCG 
                 12 
                 TAGTGTTTGCAAGCACATCG 
                 52 
                 AGAGTAGCTCACTTCCGA 
                 92 
               
               
                   
               
               
                 DYRK1A 
                 TGATTATATTGTAAAAAACG 
                 13 
                 ATCAAGCCCAGATAGAAGTG 
                 53 
                 CTCCGTGAACGTTCGTGA 
                 93 
               
               
                   
               
               
                 GFP 
                 ACCAGGATGGGCACCACCCG 
                 14 
                 ACCAGGATGGGCACCACCCG 
                 54 
                 CGAGCCTCTACTTGGCGC 
                 94 
               
               
                 (control) 
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 FBXO11 
                 ACACGCAAGCAGCTCTACAA 
                 15 
                 CCGGCGTTGTTCCGATCCTG 
                 55 
                 GGTAGTGGTGCACACACG 
                 95 
               
               
                   
               
               
                 FOXP1 
                 TGTTGAGGAGTGATAACCTG 
                 16 
                 CAACCACTTACTAGAGTGCG 
                 56 
                 CCCTAGGAATTCTTAATT 
                 96 
               
               
                   
               
               
                 GATAD2B 
                 TTGCCTCCCATATCCAACCA 
                 17 
                 CGTTGAGACATCAACATGTG 
                 57 
                 AATGTTTTCACGGTTGTT 
                 97 
               
               
                   
               
               
                 KDM5B 
                 ATTCAGCCTCTGGATCCGCG 
                 18 
                 AGACTGGGATCTGTAAGGT 
                 58 
                 CACGAGCGCAACCTCAGT 
                 98 
               
               
                   
               
               
                 LARP4B 
                 GATATCGGAGTCTACCCCCG 
                 19 
                 CAGGCACAGCGAGTCCAGGA 
                 59 
                 ATTTCATGACGCAATTTG 
                 99 
               
               
                   
               
               
                 MAP1A 
                 GCTGGTCCTATCCTCACCAG 
                 20 
                 TGTTGAACATAAGGCTCCGG 
                 60 
                 CCGCAGGTAGTGGGCTGT 
                 100 
               
               
                   
               
               
                 MBD5 
                 TCCAGTAGTACCTTCACGGG 
                 21 
                 CCATGCTCTGTAATAGACGG 
                 61 
                 CGGACAATGGAACGAGGA 
                 101 
               
               
                   
               
               
                 MED13L 
                 GTTCGCTACCCAGTTCGCCG 
                 22 
                 ACGCCATACACACAGCAGGT 
                 62 
                 GAGCTTGGTCGCAGAGTA 
                 102 
               
               
                   
               
               
                 MLL1 
                 GGATCATCAAGACTCCCCGG 
                 23 
                 AGAAAGGGCGGCGATCAAGG 
                 63 
                 AGAAAACTACATACCGCA 
                 103 
               
               
                   
               
               
                 MYST4 
                 ATTGGAATGGGATCGGCACG 
                 24 
                 CAAATGTGAAGGCCTTGAGG 
                 64 
                 CGGATGCCCGAATCACCA 
                 104 
               
               
                   
               
               
                 POGZ 
                 ATTGTGCTGAACGTACAGCA 
                 25 
                 CACTACTGTTAGTAACAGTG 
                 65 
                 CCAACGCGTCTTCTGGCC 
                 105 
               
               
                   
               
               
                 PTEN 
                 TGTGCATATTTATTGCATCG 
                 26 
                 TCACCTGGATTACAGACCCG 
                 66 
                 CCTATCTTTAGACGGATG 
                 106 
               
               
                   
               
               
                 QRICH1 
                 AGTACATCCGAGTAAAGGCG 
                 27 
                 TCCCCAGGAAGCCTACAATG 
                 67 
                 CCCGAACTGTTTCACCCA 
                 107 
               
               
                   
               
               
                 SATB2 
                 AGAGCTGTGGGAATACCCCA 
                 28 
                 CAGCCGGGCCACCTTCACCG 
                 68 
                 CCGCTTCGTGTGTCGAAT 
                 108 
               
               
                   
               
               
                 SCN2A 
                 GGGAGTTAAAATGTACAGGG 
                 29 
                 GGGATTCCCTGGTAAAGAAG 
                 69 
                 TGTGGGCGGTATGGGAGG 
                 109 
               
               
                   
               
               
                 SETD2 
                 TCTAGGTCACCTGAATCCAG 
                 30 
                 TAGAAATCCCCCATCTTCGG 
                 70 
                 CGGTTGACAGTTCGTCTG 
                 110 
               
               
                   
               
               
                 SETD5 
                 TCGACACCCATGCCTCTGAG 
                 31 
                 TCGCCCGTAGAGGAACGCTG 
                 71 
                 CAGCTTTTGCAGTTGCGG 
                 111 
               
               
                   
               
               
                 SPEN 
                 CCTATGGACACCATGAACGG 
                 32 
                 GAATCTTGACACTTTCCACG 
                 72 
                 CTTCAGCTTTGACACACA 
                 112 
               
               
                   
               
               
                 STARD9 
                 TATGAACTGGGAGATCCCTG 
                 33 
                 GCAGCTGAGGAAGCACATCG 
                 73 
                 TTAAAATGCCGCGTTTGG 
                 113 
               
               
                   
               
               
                 SUV420H1 
                 ATTACAGCAGCACTCGGGCA 
                 34 
                 CTCCTTGGCGGACATTCCAG 
                 74 
                 CAGTGCTACACGGTTGCC 
                 114 
               
               
                   
               
               
                 SYNGAP1 
                 AGGGGGCATAGGACATCGCG 
                 35 
                 CCAGCCAGGACGATCGTACG 
                 75 
                 CCGGCAGGGGAATACGTG 
                 115 
               
               
                   
               
               
                 TCF20 
                 AGAGCTATGGACCTCCCCAG 
                 36 
                 ATCAAACATGAGACTTACCG 
                 76 
                 CTAATCGGGTTTCGGCTT 
                 116 
               
               
                   
               
               
                 TCF7L2 
                 GTGTACCCAATCACGACAGG 
                 37 
                 CGGAAACTTTCGGAGCGAGG 
                 77 
                 TTGTATCGTAGGTCATCA 
                 117 
               
               
                   
               
               
                 TNRC6B 
                 ATAAAGTGTTACTAAAACGT 
                 38 
                 TGTTCCCATGCAAACCAATG 
                 78 
                 CTTGCACATGTTGGGAGA 
                 118 
               
               
                   
               
               
                 UPF3B 
                 CGATAGGCAGGATCGCAACA 
                 39 
                 TGTTCCTTGGTCAAAGTGGG 
                 79 
                 AACCTTTATTTGGCGCCG 
                 119 
               
               
                   
               
               
                 WAC 
                 TGACAGCACAGGTCACAACA 
                 40 
                 TTGAACTATGAAGTGCACTG 
                 80 
                 CTGGTACAAGGCGTAGAT 
                 120 
               
               
                   
               
            
           
         
       
     
     In Vivo Perturb-Seq Experiment 
     The backbone plasmid contains antiparallel cassettes of two gRNAs (Table 6) under mouse U6 and human U6 promoters, and the EF1a promoter to express puromycin, BFP, and a polyadenylated barcode unique to each perturbation. Cloning and lentiviral packaging of the 38 vectors were done individually. 
     All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committees (IACUC) of Harvard University and of the Broad Institute of MIT and Harvard. In utero lentiviral injection into the lateral ventricles was performed at E12.5 in Cas9 transgenic mice (14) (4-6 month old, Jax #026179), and each single-cell library was made by combining the BFP+ cells from 1-3 litters (4-20 animals) of P7 animals harvested on the same day. Tissue dissociation was performed with the Papain Dissociation kit (Worthington, #LK003152). The FACS-purified cells were sorted into cold Hibernate A/B27 medium and subjected to single-cell RNA sequencing library preparation. This analysis included 17 independent libraries of Perturb-Seq cells. 
     Single-cell RNA sequencing libraries were created using the Chromium Single Cell 3′ Solution v2 kit (10× Genomics) following the manufacturer&#39;s protocol. Each library was sequenced with Illumina NextSeq high-output 75-cycle kit with sequencing saturation above 70%. Dial-out PCR was performed to extract the perturbation barcode in each cell. 
     Perturbation barcodes were identified by two complementary methods. The dial-out sequences were first used to create a cell-by-perturbation UMI count matrix by a modification of from the original Perturb-Seq work (12). In addition, barcode sequences were extracted from the 10× Genomics Cell Ranger bam file. Reads were then assigned to the perturbation they mapped best. Cell barcodes and UMIs were extracted, and a cell-by-perturbation UMI count matrix was created. Cells for which either i) the assigned 10× and dialout perturbations agree or ii) the cell was assigned to a perturbation by one method but not assigned to any perturbation in the other were then kept. 
     This analysis comprises 17 independent libraries of Perturb-Seq cells. In utero lentiviral injection into the lateral ventricles was performed at E12.5 in Cas9 transgenic mice (14) (4-6 month old, Jax #026179), and each 10× single-cell library was made by combining the BFP+ cells from 1-3 litters (4-20 animals) of P7 animals harvested on the same day. P7 mice were anesthetized then disinfected with 70% ethanol and decapitated. The brains were quickly extracted into ice-cold PBS and cortices were micro-dissected in ice-cold Hibernate A medium (BrainBits, #HA-Lf) with B27 supplement (ThermoFisher, #17504044) under a dissecting microscope. Tissue dissociation was performed with the Papain Dissociation kit (Worthington, #LK003152) in a modification of a previously described protocol (50). Briefly, cortices were transferred into ice-cold papain solution with DNase in a cell culture dish and cut into small pieces with a blade. The dish was then placed onto a digital rocker in a cell culture incubator for 30 mins with rocking speed at 30 rpm at 37° C. The digested tissues were collected into a 15 mL tube with 5 mL of EBSS buffer (from the Worthington kit). The mixture was triturated with a 10 mL plastic pipette 20 times and the cell suspension was carefully transferred to a new 15 mL tube. 2.7 mL of EBSS, 3 mL of reconstituted Worthington inhibitor solution, and DNAse solution were added to the 15 mL tube and mixed gently. Cells were pelleted by centrifugation at 300 g for 5 mins at RT. Cells were resuspended in 0.5 mL ice-cold Hibernate A with B27 supplement (ThermoFisher, A3582801) and 10% fetal bovine serum (FBS) and subjected to FACS purification. The FACS collected cells were sorted in cold Hibernate A/B27 medium with 10% FBS (VWR, #97068). After collection, the cells were centrifuged and resuspended in ice-cold PBS with 0.04% BSA (NEB, B9000S) for single-cell RNA sequencing library preparation (10× Genomics v2 chemistry). The FACS purification and resuspension was performed within 1.5 h while keeping the cells on ice to prevent necrosis. 
     Perturb-Seq Analysis 
     UMI count data was loaded into R and processed using the Seurat v 2.2 package (47). Clusters were assigned to cell types based on marker genes from the literature, mousebrain.org (16), and DropViz.com (22). Only cells of 5 key types (projection neurons, inhibitory neurons, oligodendrocytes, microglia/macrophages, and astroglia) were focused on and the rest were removed. WGCNA and Structural topic modelling (STM) were performed for each cell cluster based on the published pipelines (20, 21). Linear regression was used to test the relationship between perturbations and WGCNA gene scores, correcting for batch and number of genes. To test for correlations between perturbations and topics, the theta matrix (the matrix containing proportions of topics per cell) was extracted from the STM matrix. For each topic, linear regression was used to test how the per-cell proportions for each topic related to perturbations (after setting GFP to be the reference perturbation), correcting for nGene and batch. 
     RNA In Situ Hybridization and Immunohistochemistry 
     Multiplexed RNAscope fluorescent in situ hybridization and immunohistochemistry was performed on fixed-frozen tissue. Mice were anesthetized and transcardially perfused with ice-cold PBS followed by ice-cold 4% paraformaldehyde in PBS. Dissected brains were postfixed overnight in 4% paraformaldehyde at 4° C., and cryoprotected in 30% sucrose. Brains were then embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, #4583) and 15-20 pm tissue sections were prepared. 
     Multiplex RNAscope v1 was performed based on manufacturer&#39;s instructions. Probes against the following mRNA were used: Pdgfra, Cspg4, and Fezf2 (ACDBio). The staining, imaging, and quantifications were double-blinded. Quantification was performed using the StarSearch program (https://www.seas.upenn.edu/˜rajlab/StarSearch/launch.html). 
     For immunohistochemistry, mice were anesthetized and transcardially perfused with ice-cold PBS followed by ice-cold 4% paraformaldehyde in PBS. Dissected brains were postfixed overnight in 4% paraformaldehyde at 4° C., and cryoprotected in 30% sucrose. The brains were embedded in OCT compound (Tissue-Tek, #4583) and 15 μm tissue sections were prepared. The slides with tissue sections were incubated with blocking media (6% donkey serum in 0.3% Triton with PBS) for 1 hr, then incubated with primary antibodies in the incubation media (1:3 dilution of blocking media in PBS with 0.3% Triton) overnight at 4° C. Slides were washed with PBS with 0.3% Triton 4 times to remove the excess primary antibody. Secondary antibodies were applied at 1:800 dilution in blocking media and incubated for 2 hr at room temperature. Slides were then washed 4 times with PBS with 0.3% Triton and incubated with DAPI for 10 mins before mounting with Fluoromount G (Invitrogen, #00-4958-02). The antibodies and dilutions were: Mouse anti-NeuN antibody (mab377, 1:500; Millipore), Mouse anti-GS antibody (mab302, 1:500; Millipore), Goat anti-Pdgfra antibody (AF1062, 1:200; R&amp;D System), Rabbit Ibal antibody (019-19741, 1:400; Wako), Chicken anti-GFP antibody (ab16901, 1:500; Millipore), Mouse anti-Satb2 (ab51502, 1:50; Abcam), Rat anti-Ctip2 (ab18465, 1:100, Abcam), Rabbit anti-Sox6 (ab30455, 1:500; Abcam), Rat anti-Mbp (mab386, 1:100; Millipore). 
     All images were acquired using either a custom-built spinning disk confocal microscope equipped with image acquisition NIS-Elements software, or a Carl Zeiss epifluorescent microscope with Zen software. To quantify protein expression levels, the thickness of the cortex was divided into bins and calculated the average pixel value per bin was calculated. The staining, imaging, and quantifications were double-blinded. 
     Perturb-Seq Profiling 
     Single-cell RNA sequencing libraries were created using the Chromium Single Cell 3′ Solution v2 kit (10× Genomics) following the manufacturer&#39;s protocol. Each library was sequenced with Illumina NextSeq high-output 75-cycle kit with sequencing saturation above 70%. Reads were aligned to the mm10 mouse genome reference using the Cell Ranger package (10× Genomics). 
     To sequence the perturbation barcode, dial-out PCR was performed to extract the perturbation barcode in each cell. This is modified from Dixit et al (12) to be compatible with the 10× Genomic V2 chemistry instead of V1. The PCR product was sequenced along with the 10× libraries, and demultiplexed to extract the perturbation information. 
     
       
         
           
               
               
            
               
                   
                 Forward primer: 
               
               
                   
                 (SEQ ID NO: 121) 
               
               
                   
                 CAAGCAGAAGACGGCATACGAGAT-TCGCCTTA- 
               
               
                   
               
               
                   
                 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG- 
               
               
                   
               
               
                   
                 TAGCAAACTGGGGCACAAGC 
               
               
                   
               
               
                   
                 Reverse primer (i5): 
               
               
                   
                 (SEQ ID NO: 122) 
               
               
                   
                 AATGATACGGCGACCACCGAGATCTACAC 
               
            
           
         
       
     
     Data Analysis 
     Data Pre-Processing 
     BCL files were transformed into fastq files using the cellranger mkfastq command, using CellRanger V2.1.0. Bam files and expression matrices were generated from these fastq files using the cellranger count command, using force cells=8000. 
     Identification of Perturbation Barcode 
     Perturbation barcodes were identified by two complementary methods. To extract perturbation information from the dial-out reads, code was modified from the original Perturb-Seq work (12) to work with 10× V2 chemistry and was applied to the data (original code at https://github.com/asncd/MIMOSCA). This resulted in a cell-by-perturbation UMI count matrix. To extract perturbation information from the 10× reads, a fasta file was first created with one entry for each perturbation, containing the sequence of the perturbation barcode and the surrounding sequence. This fasta file was turned into a STAR reference (51), referred to as the PBC reference. Unmapped reads containing either AGAATT or CCTAGA as a subsequence were extracted from the Cell Ranger bam file, and then mapped to this new reference. Low quality reads were filtered out using the following filters: (i) used “samtools view -F 2820” to filter out unmapped, multimapped, and low quality reads from the PBC mapped bam file, (ii) removed reads with quality scores &lt;255 (iii) removed reads whose 5′ end did not map between 655 and 714 bp into the PBC reference, to help exclude reads that did not overlap enough bases in the perturbation barcode for proper identification of the perturbation, and (iv) removed reads whose edit distance from the PBC reference was &gt;2. Reads were then assigned to the perturbation they mapped best. Cell barcodes and UMIs were extracted, and a cell-by-perturbation UMI count matrix was created. This matrix was used to assign cells to perturbations in the same way as with the dial-out data. As with the dialout data, if a cell had one perturbation with &gt;1.3× the number of UMIs assigned to it than the next best perturbation based on the 10× sequence, that cell was assigned to that perturbation in the 10× data; otherwise, the cell was declared to have multiple perturbations. Only cells for which either i) the assigned 10× and dialout perturbations agree or ii) the cell was assigned to a perturbation by one method but not assigned to any perturbation in the other were kept. 
     Cell Type Clustering Analysis 
     UMI count data was loaded into R and processed using the Seurat v 2.2 package (47). Data were scaled to counts per million and log normalized. Cells expressing less than 500 genes were removed. Variable genes were found using FindVariableGenes with x.low.cutoff=1 for each batch separately. Genes that were found to be variable in at least 4 batches were combined into a final combined list of variable genes. The normalized data was scaled with ScaleData on the variable genes, regressing out the effects of nUMI, and PCA was performed. Clustering was performed with the FindClusters function (with default parameters, except for resolution=1.2 and using 28 PCs). tSNE plots were generated with RunTSNE (RunTSNE (with default parameters, except with 17 PCs and pca=F). Clusters were assigned to cell types based on marker genes from the literature, mousebrain.org (16), and DropViz (22). For each cell type, a more refined nGene cutoff was identified ( FIGS. 7A-7G ), and cells of that cell type with less than that filter were removed from further consideration. Cell clustering does not follow the proportion of mitochondrial reads or nUMI in each cell. Only cells of 5 key types (projection neurons, inhibitory neurons, oligodendrocytes, microglia/macrophages, and astroglia) were focused on and the rest were removed. 
     For subclustering individual cell types, the cells of that cell type were extracted from the larger Seurat object. Variable genes were chosen as above, and data was scaled with ScaleData, regressing out the effects of nUMI and batch, followed by PCA. Clustering was performed with FindClusters (with default parameters except for varying resolutions and number of PCs, Table 7). tSNE was performed with RunTSNE (with default parameters, except with different numbers of PCs and pca=F). 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Parameters used in Seurat for cell type clustering. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Required 
                   
               
               
                 Dataset 
                 #PCs Used 
                 Resolution Used 
                 Variable Genes 
                 Batches*** 
                 Note 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 Cortex Perturb-seq 
                 28 
                 1.2 
                 Calculated on each batch and 
                 4 
                   
               
               
                   
                   
                   
                 combined** 
               
               
                 Striatium Perturb-seq 
                 22 
                 1.2 
                 Calculated on each batch and 
                 2 
               
               
                   
                   
                   
                 combined** 
               
               
                 Single Perturb ANK2 and WT 
                 11 
                 0.8 
                 Jointly calculated* 
                 NA 
               
               
                 joint 
               
               
                 WT P7 Dataset 
                 15 
                 0.8 
                 Jointly calculated* 
                 NA 
               
               
                 10X E18.5 Dataset 
                 13 
                 0.8 
                 Jointly calculated* 
                 NA 
                 Publicly 
               
               
                   
                   
                   
                   
                   
                 available data 
               
            
           
           
               
            
               
                 Cortex CellTypes 
               
               
                 Subclustering 
               
            
           
           
               
               
               
               
               
               
            
               
                 Astroglia 
                 15 
                 0.5 
                 Calculated on each batch and 
                 4 
                   
               
               
                   
                   
                   
                 combined** 
               
               
                 Inhibitory Neurons 
                 11 
                 0.8 
                 Calculated on each batch and 
                 4 
               
               
                   
                   
                   
                 combined** 
               
               
                 Excitatory Neurons 
                 15 
                 0.8 
                 Calculated on each batch and 
                 4 
               
               
                   
                   
                   
                 combined** 
               
               
                 Microglia 
                 11 
                 0.3 
                 Calculated on each batch and 
                 4 
               
               
                   
                   
                   
                 combined** 
               
               
                 ODC 
                 10 
                 0.5 
                 Calculated on each batch and 
                 4 
               
               
                   
                   
                   
                 combined** 
               
               
                   
               
            
           
         
       
     
     Testing WGCNA Gene Sets 
     WGCNA was performed for each cell cluster based on the published pipeline (21). Modules that were driven by outlier cells (these are modules that are highly expressed in a very small number of cells; this is the module level quality control, equivalent of filtering out genes expressed in a small number of cells) were manually removed. For a given cell type, each WGCNA gene set was input into moduleEigengenes to calculate a gene-set score for that set of genes. All cells without an assigned perturbation were removed. 
     Linear regression was used to test the relationship between perturbations and WGCNA gene scores, correcting for batch and number of genes with the lm function in R, using the formula: 
       Gene Score ˜perturbation+batch+nGene
 
     Associated P-values and effect sizes were extracted. In addition, a permutation-based approach was used to calculate an empirical P-value to ensure the model-based P-values reported by lm were accurate. Specifically, the perturbation labels of cells were randomly permuted within each batch, and the absolute effect size for each perturbation was calculated as above on this permuted data. This was repeated 10,000 times. The empirical P-value was the proportion of permutations (including the original data) with absolute effect size larger than that of the original data. FDR correction was performed using the Benjamini &amp; Hochberg procedure. 
     To implement alternative analytical assumptions that do not rely on individual cells being independent conditional on batch, a linear mixed model-based approach was used. The lmer function from the lmerTest package in R was used. For each module, used this function was used to fit a linear mixed model of the scaled module scores with random interaction effect for each batch/perturbation pair, and fixed effects for batch, perturbation, and scaled nGene. This was performed with the R formula: 
       WGCNA_Score ˜batch+perturbation+nGene+(1|batch:perturbation)
 
     where WGCNA Score and nGene were mean centered and normalized to have variance 1. The p-values and effect sizes for each perturbation were then extracted from the resulting model. 
     Structural Topic Modelling 
     Structural topic modelling (STM) was performed separately on each cell type of interest using the STM package in R (20). Count data from cells of a given type were extracted from the Seurat object, along with corresponding meta data. Genes that occurred in &lt;5% or &gt;90% of cells were removed, as were mitochondrial and ribosomal genes. In addition, only genes that were expressed in at least one cell in all batches were retained in order to help reduce batch effects. The resulting count matrix was provided as input to the STM function, along with the meta data and with parameters LDAbeta=T, interactions=F. The formula used by the STM function was 
       ˜perturbation+batch+nGene
 
     This specifies a model that assumed topic proportions were dependent on perturbation, number of genes, and batch. This model was run on each dataset with 5 topics. Top 10 genes for each topic were extracted with the labelTopics function. 
     To test for correlations between perturbations and topics, the theta matrix (the matrix containing proportions of topics per cell) was extracted from the STM matrix. For each topic, linear regression was used to test how the per-cell proportions for each topic related to perturbations (after setting GFP to be the reference perturbation), correcting for nGene and batch. In particular, the lm function in R was used, with the formula: 
       Proportion Topic ˜perturbation+batch+nGene
 
     Effect sizes were extracted from the resulting lm object. An empirical P-value was calculated, as for WGCNA. FDR correction was performed using the Benjamini &amp; Hochberg procedure. 
     Correlation Graph of WGCNA Genes 
     For each cell type, all genes that appeared in at least one module for that cell type were extracted and the correlation between each pair was calculated. An 11 nearest neighbor graph was constructed, and the results were plotted with the igraph (v1.2.4.1) plot feature. 
     Analysis of Human Single Nucleus or Single Cell RNA-Seq Data 
     For each single cell/nucleus human dataset, the UMI count matrix and meta data were downloaded (adult human data: https://portal.brain-map.org/atlases-and-data/rnaseq/human-multiple-cortical-areas-smart-seq/, fetal human data: https://cortex-dev.cells.ucsc.edu/, human cerebral organoids data: https://singlecell.broadinstitute.org/singlecell/study/SCP282/reproducible-brain-organoids/) and processed with Seurat to create Seurat objects, with no nGene cutoff. Cell types were extracted from the metadata, and combined into more general cell types, namely: Microglia, Astroglia (including Radial Glia), Inhibitory neurons, and Excitatory neurons, ODCs, and others. Correlation analysis was then performed on these data as described in the ‘Correlation Analysis’ section. 
     Gene Module Conservation and Modularity: Correlation Analysis 
     For each dataset and each module, the associated cell type was extracted. The number of genes in the module expressed in at least 1% or 5% of cells were calculated. All genes expressed in &lt;5% of cells were then excluded, as were modules with &lt;3 genes surviving this 5% cutoff. The Pearson correlation coefficient between each pair of genes in the module was calculated, and the mean of these coefficients was calculated. For each module, a null distribution of the mean correlation coefficient was calculated as follows: a random set of genes was chosen with the same number of genes as the WGCNA module and roughly the same expression levels (all genes expressed by that cell type were partitioned into 100 mean expression bins, and randomly sampled genes from the matched bin for each gene in the module), and the average correlation coefficient was calculated as above. This was repeated 1,000 times, and an empirical P-value was estimated as the proportion of gene sets with correlation greater than that in the WGCNA module, as was an expected value for this average correlation coefficient. The normalized correlation was calculated by dividing the average correlation of the WGCNA module by the standard deviation of the correlation value from the matching null distribution and subtracting the mean correlation. Confidence intervals were calculated using bootstrapping (boot package v1.3-20 in R). For human single nucleus RNA-seq data, genes in each module were mapped to 1:1 human orthologs (from BioMart), before performing the above analysis. 
     Analysis of Human Bulk Data 
     Bulk human RNA-seq data was downloaded from BrainSpan (https://www.brainspan.org/static/download.html) and log transformed. For each module, the average expression of the genes of that module were calculated, and the results were plotted. 
     Differential Expression Analysis 
     For each cell type, raw count data was extracted, and genes expressed in &lt;5% of cells were removed. linear v3.36.2 (52) was then used to perform differential expression analysis, fitting a linear model for each gene with batch and perturbation as covariates. For each perturbation, the associated P-value and log FC relative to GFP was calculated, followed by FDR correction. Results are shown in Table 8. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Analysis of differential gene expression from Perturb-Seq data. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Estimate 
                 SE 
                 Z 
                 pvalue 
                 Gene 
                 Condition 
                 padj 
                 CellType 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 2.64461235 
                 0.06330411 
                 41.7763105 
                 0 
                 Plp1 
                 Ddx3x 
                 0 
                 ODC 
               
               
                 2.2130509 
                 0.06262683 
                 35.3371042 
                  1.58E−273 
                 Plp1 
                 Upf3b 
                  1.27E−267 
                 ODC 
               
               
                 2.14029504 
                 0.06290119 
                 34.0263044 
                  9.10E−254 
                 Plp1 
                 Setd5 
                  4.87E−248 
                 ODC 
               
               
                 2.12907122 
                 0.06672781 
                 31.906806 
                  2.15E−223 
                 Plp1 
                 Setd2 
                  8.63E−218 
                 ODC 
               
               
                 1.96778043 
                 0.06277633 
                 31.3458988 
                  1.11E−215 
                 Plp1 
                 Fbxo11 
                  3.56E−210 
                 ODC 
               
               
                 2.01760549 
                 0.06564918 
                 30.7331398 
                  2.05E−207 
                 Plp1 
                 Myst4 
                  5.50E−202 
                 ODC 
               
               
                 1.83789934 
                 0.06300006 
                 29.1729795 
                  4.27E−187 
                 Plp1 
                 Qrich1 
                  9.80E−182 
                 ODC 
               
               
                 1.80246643 
                 0.06306056 
                 28.5831012 
                  1.09E−179 
                 Plp1 
                 Wac 
                  2.19E−174 
                 ODC 
               
               
                 1.77653611 
                 0.06260943 
                 28.374895 
                  4.13E−177 
                 Plp1 
                 Kdm5b 
                  7.37E−172 
                 ODC 
               
               
                 1.79975815 
                 0.06414067 
                 28.0595487 
                  3.05E−173 
                 Plp1 
                 Stard9 
                  4.91E−168 
                 ODC 
               
               
                 1.84320151 
                 0.06636854 
                 27.7722182 
                  9.40E−170 
                 Plp1 
                 Adnp 
                  1.37E−164 
                 ODC 
               
               
                 1.19892624 
                 0.04843699 
                 24.7522878 
                  2.93E−135 
                 Mbp 
                 Upf3b 
                  3.92E−130 
                 ODC 
               
               
                 1.3717565 
                 0.05738902 
                 23.9027712 
                  2.87E−126 
                 Mbp 
                 Gatad2b 
                  3.54E−121 
                 ODC 
               
               
                 1.53098402 
                 0.06433277 
                 23.7978856 
                  3.51E−125 
                 Plp1 
                 Cul3 
                  4.03E−120 
                 ODC 
               
               
                 1.27727929 
                 0.05500932 
                 23.2193234 
                  2.91E−119 
                 Plp1 
                 Dyrk1a 
                  3.11E−114 
                 ODC 
               
               
                 1.40796073 
                 0.06522523 
                 21.5861381 
                  2.42E−103 
                 Plp1 
                 Ctnnb1 
                 2.43E−98 
                 ODC 
               
               
                 1.08085901 
                 0.0502152 
                 21.5245392 
                  9.17E−103 
                 Mbp 
                 Ddx3x 
                 8.67E−98 
                 ODC 
               
               
                 1.04166875 
                 0.04924189 
                 21.1541182 
                 2.53E−99 
                 Mbp 
                 Setd5 
                 2.26E−94 
                 ODC 
               
               
                 1.1761105 
                 0.06569099 
                 17.9036811 
                 1.10E−71 
                 Plp1 
                 Asxl3 
                 9.33E−67 
                 ODC 
               
               
                 0.91407848 
                 0.05356213 
                 17.0657594 
                 2.67E−65 
                 Mbp 
                 Myst4 
                 2.14E−60 
                 ODC 
               
               
                 0.84874703 
                 0.04994379 
                 16.9940443 
                 9.09E−65 
                 Mbp 
                 Stard9 
                 6.95E−60 
                 ODC 
               
               
                 1.95714956 
                 0.11578085 
                 16.9039144 
                 4.21E−64 
                 Hapln1 
                 Dscam 
                 3.07E−59 
                 Inhibitory 
               
               
                 1.08598415 
                 0.06523423 
                 16.6474577 
                 3.16E−62 
                 Plp1 
                 Tnrc6b 
                 2.21E−57 
                 ODC 
               
               
                 0.87345148 
                 0.05276075 
                 16.5549478 
                 1.47E−61 
                 Mbp 
                 Adnp 
                 9.87E−57 
                 ODC 
               
               
                 2.00154666 
                 0.12396699 
                 16.1458041 
                 1.22E−58 
                 Hapln1 
                 Pogz 
                 7.81E−54 
                 Inhibitory 
               
               
                 1.850412 
                 0.11582849 
                 15.9754482 
                 1.89E−57 
                 Hapln1 
                 Setd5 
                 1.17E−52 
                 Inhibitory 
               
               
                 0.6100828 
                 0.03968568 
                 15.3728695 
                 2.49E−53 
                 Mbp 
                 Dyrk1a 
                 1.48E−48 
                 ODC 
               
               
                 1.76991222 
                 0.11589322 
                 15.2719218 
                 1.18E−52 
                 Hapln1 
                 Asxl3 
                 6.75E−48 
                 Inhibitory 
               
               
                 1.76803931 
                 0.11730127 
                 15.0726353 
                 2.45E−51 
                 Cldn11 
                 Ddx3x 
                 1.36E−46 
                 ODC 
               
               
                 0.81378625 
                 0.05433407 
                 14.9774568 
                 1.03E−50 
                 Mbp 
                 Setd2 
                 5.52E−46 
                 ODC 
               
               
                 2.12131543 
                 0.14226301 
                 14.9112224 
                 2.79E−50 
                 Hapln1 
                 Larp4b 
                 1.44E−45 
                 Inhibitory 
               
               
                 1.65988811 
                 0.11307849 
                 14.6790796 
                 8.78E−49 
                 Hapln1 
                 Stard9 
                 4.41E−44 
                 Inhibitory 
               
               
                 1.73764682 
                 0.12109719 
                 14.3491922 
                 1.08E−46 
                 Hapln1 
                 Cul3 
                 5.25E−42 
                 Inhibitory 
               
               
                 1.79524068 
                 0.12644001 
                 14.1983588 
                 9.38E−46 
                 Hapln1 
                 Syngap1 
                 4.43E−41 
                 Inhibitory 
               
               
                 −2.7570451 
                 0.20155739 
                 −13.67871 
                 1.36E−42 
                 Mbp 
                 Med13l 
                 6.25E−38 
                 ODC 
               
               
                 1.56334928 
                 0.1148096 
                 13.6168861 
                 3.18E−42 
                 Cldn11 
                 Upf3b 
                 1.42E−37 
                 ODC 
               
               
                 0.68475929 
                 0.05036527 
                 13.5958615 
                 4.24E−42 
                 Mbp 
                 Cul3 
                 1.84E−37 
                 ODC 
               
               
                 1.65812423 
                 0.12269634 
                 13.5140484 
                 1.29E−41 
                 Hapln1 
                 Setd2 
                 5.46E−37 
                 Inhibitory 
               
               
                 1.122117 
                 0.08502534 
                 13.1974415 
                 9.08E−40 
                 Plp1 
                 Gatad2b 
                 3.74E−35 
                 ODC 
               
               
                 1.13794128 
                 0.08704476 
                 13.0730593 
                 4.69E−39 
                 Hapln1 
                 Mll1 
                 1.89E−34 
                 Inhibitory 
               
               
                 −0.9472644 
                 0.07354873 
                 −12.879412 
                 5.88E−38 
                 Mbp 
                 Spen 
                 2.30E−33 
                 ODC 
               
               
                 1.52734574 
                 0.11876674 
                 12.8600464 
                 7.55E−38 
                 Hapln1 
                 Scn2a1 
                 2.89E−33 
                 Inhibitory 
               
               
                 1.51656812 
                 0.11840093 
                 12.8087523 
                 1.46E−37 
                 Hapln1 
                 Tcf20 
                 5.47E−33 
                 Inhibitory 
               
               
                 1.77633679 
                 0.14065845 
                 12.6287247 
                 1.47E−36 
                 Hapln1 
                 Ank2 
                 5.35E−32 
                 Inhibitory 
               
               
                 3.58245737 
                 0.2854763 
                 12.5490536 
                 4.02E−36 
                 Hbb−bs 
                 Setd2 
                 1.44E−31 
                 Excitatory 
               
               
                 0.62038203 
                 0.04981951 
                 12.452591 
                 1.35E−35 
                 Mbp 
                 Wac 
                 4.73E−31 
                 ODC 
               
               
                 0.90249648 
                 0.07254447 
                 12.4405965 
                 1.57E−35 
                 Plp1 
                 Tcf20 
                 5.38E−31 
                 ODC 
               
               
                 1.52285969 
                 0.1239567 
                 12.2854161 
                 1.08E−34 
                 Cldn11 
                 Setd2 
                 3.63E−30 
                 ODC 
               
               
                 1.47879536 
                 0.12070608 
                 12.2512081 
                 1.66E−34 
                 Hapln1 
                 Fbxo11 
                 5.43E−30 
                 Inhibitory 
               
               
                 0.71217798 
                 0.05845683 
                 12.1829727 
                 3.83E−34 
                 Plp1 
                 Mll1 
                 1.23E−29 
                 ODC 
               
               
                 1.54224288 
                 0.12984273 
                 11.8777764 
                 1.54E−32 
                 Hapln1 
                 Spen 
                 4.86E−28 
                 Inhibitory 
               
               
                 1.37538193 
                 0.11585407 
                 11.8716759 
                 1.66E−32 
                 Cldn11 
                 Setd5 
                 5.13E−28 
                 ODC 
               
               
                 0.57932531 
                 0.04984933 
                 11.621527 
                 3.20E−31 
                 Mbp 
                 Kdm5b 
                 9.71E−27 
                 ODC 
               
               
                 1.42703337 
                 0.12894857 
                 11.0666865 
                 1.82E−28 
                 Hapln1 
                 Med13l 
                 5.41E−24 
                 Inhibitory 
               
               
                 1.29215236 
                 0.11876542 
                 10.8798703 
                 1.44E−27 
                 Hapln1 
                 Qrich1 
                 4.20E−23 
                 Inhibitory 
               
               
                 1.51809103 
                 0.14395929 
                 10.5452801 
                 5.34E−26 
                 Hapln1 
                 Ddx3x 
                 1.53E−21 
                 Inhibitory 
               
               
                 −1.5248064 
                 0.14620497 
                 −10.429238 
                 1.82E−25 
                 Mbp 
                 Larp4b 
                 5.14E−21 
                 ODC 
               
               
                 −1.2795607 
                 0.12425109 
                 −10.298185 
                 7.18E−25 
                 Mbp 
                 Chd8 
                 1.99E−20 
                 ODC 
               
               
                 1.33145689 
                 0.12995055 
                 10.245873 
                 1.24E−24 
                 Hapln1 
                 Pten 
                 3.36E−20 
                 Inhibitory 
               
               
                 4.0057396 
                 0.39502809 
                 10.1403917 
                 3.66E−24 
                 Ptgds 
                 Satb2 
                 9.79E−20 
                 Astroglia 
               
               
                 1.17327934 
                 0.11793573 
                 9.94846418 
                 2.56E−23 
                 Cldn11 
                 Stard9 
                 6.74E−19 
                 ODC 
               
               
                 1.19524848 
                 0.12301362 
                 9.71639138 
                 2.57E−22 
                 Cldn11 
                 Myst4 
                 6.65E−18 
                 ODC 
               
               
                 1.17485963 
                 0.12253383 
                 9.58804264 
                 8.98E−22 
                 Hapln1 
                 Upf3b 
                 2.29E−17 
                 Inhibitory 
               
               
                 3.62215404 
                 0.37799721 
                 9.58248872 
                 9.47E−22 
                 Hba-a1 
                 Setd2 
                 2.38E−17 
                 Excitatory 
               
               
                 0.47847783 
                 0.05045429 
                 9.48339182 
                 2.46E−21 
                 Mbp 
                 Qrich1 
                 6.08E−17 
                 ODC 
               
               
                 0.4792329 
                 0.05069892 
                 9.45252619 
                 3.31E−21 
                 Mbp 
                 Fbxo11 
                 8.05E−17 
                 ODC 
               
               
                 −0.6366838 
                 0.06803036 
                 −9.35882 
                 8.06E−21 
                 Mbp 
                 Ash1l 
                 1.93E−16 
                 ODC 
               
               
                 1.05911863 
                 0.11615273 
                 9.11832755 
                 7.63E−20 
                 Cldn11 
                 Wac 
                 1.80E−15 
                 ODC 
               
               
                 1.15204294 
                 0.12913762 
                 8.92104812 
                 4.62E−19 
                 Hapln1 
                 Mbd5 
                 1.08E−14 
                 Inhibitory 
               
               
                 0.37270521 
                 0.04220241 
                 8.83137225 
                 1.03E−18 
                 Mbp 
                 Mll1 
                 2.37E−14 
                 ODC 
               
               
                 −3.3661644 
                 0.38131941 
                 −8.8276765 
                 1.07E−18 
                 Mbp 
                 Pten 
                 2.42E−14 
                 ODC 
               
               
                 0.91336914 
                 0.10500124 
                 8.69865062 
                 3.36E−18 
                 Plp1 
                 Chd2 
                 7.49E−14 
                 ODC 
               
               
                 1.02071367 
                 0.11895235 
                 8.58086139 
                 9.42E−18 
                 Hapln1 
                 Tnrc6b 
                 2.07E−13 
                 Inhibitory 
               
               
                 0.99262981 
                 0.11644296 
                 8.52460096 
                 1.53E−17 
                 Cldn11 
                 Fbxo11 
                 3.33E−13 
                 ODC 
               
               
                 0.99142087 
                 0.11639021 
                 8.51807806 
                 1.62E−17 
                 Cldn11 
                 Qrich1 
                 3.47E−13 
                 ODC 
               
               
                 −2.8425153 
                 0.33891973 
                 −8.3869869 
                 4.99E−17 
                 Plp1 
                 Med13l 
                 1.05E−12 
                 ODC 
               
               
                 0.9872915 
                 0.1179803 
                 8.36827389 
                 5.85E−17 
                 Cldn11 
                 Cul3 
                 1.21E−12 
                 ODC 
               
               
                 1.37599853 
                 0.16444689 
                 8.36743397 
                 5.89E−17 
                 Hapln1 
                 Gatad2b 
                 1.21E−12 
                 Inhibitory 
               
               
                 −3.167479 
                 0.38076053 
                 −8.3188219 
                 8.88E−17 
                 Mbp 
                 Arid1b 
                 1.81E−12 
                 ODC 
               
               
                 −2.1018302 
                 0.25505653 
                 −8.2406446 
                 1.71E−16 
                 Cldn11 
                 Spen 
                 3.44E−12 
                 ODC 
               
               
                 1.04359046 
                 0.13041485 
                 8.0020831 
                 1.22E−15 
                 Hapln1 
                 Kdm5b 
                 2.43E−11 
                 Inhibitory 
               
               
                 1.35514731 
                 0.17000064 
                 7.97142494 
                 1.57E−15 
                 Hapln1 
                 Tcf7l2 
                 3.07E−11 
                 Inhibitory 
               
               
                 −2.9980472 
                 0.38080679 
                 −7.8728826 
                 3.47E−15 
                 Mbp 
                 Ank2 
                 6.71E−11 
                 ODC 
               
               
                 1.77632847 
                 0.22580299 
                 7.86671794 
                 3.64E−15 
                 Arpc1b 
                 Upf3b 
                 6.96E−11 
                 ODC 
               
               
                 1.09290491 
                 0.13903834 
                 7.86045699 
                 3.83E−15 
                 Hapln1 
                 Chd8 
                 7.23E−11 
                 Inhibitory 
               
               
                 0.39416809 
                 0.0508256 
                 7.75530564 
                 8.81E−15 
                 Mbp 
                 Tnrc6b 
                 1.63E−10 
                 ODC 
               
               
                 1.17976347 
                 0.15212681 
                 7.7551317 
                 8.83E−15 
                 Hapln1 
                 Satb2 
                 1.63E−10 
                 Inhibitory 
               
               
                 0.80871575 
                 0.10433448 
                 7.75118373 
                 9.10E−15 
                 Cldn11 
                 Dyrk1a 
                 1.66E−10 
                 ODC 
               
               
                 1.04939437 
                 0.1369567 
                 7.66223462 
                 1.83E−14 
                 Hapln1 
                 Wac 
                 3.30E−10 
                 Inhibitory 
               
               
                 0.8747889 
                 0.11630509 
                 7.52150145 
                 5.42E−14 
                 Cldn11 
                 Kdm5b 
                 9.67E−10 
                 ODC 
               
               
                 0.80201329 
                 0.10727561 
                 7.47619396 
                 7.65E−14 
                 Cldn11 
                 Mll1 
                 1.35E−09 
                 ODC 
               
               
                 0.94274224 
                 0.12716768 
                 7.41337931 
                 1.23E−13 
                 Cldn11 
                 Adnp 
                 2.15E−09 
                 ODC 
               
               
                 0.67748142 
                 0.09347785 
                 7.24750788 
                 4.25E−13 
                 Hapln1 
                 Dyrk1a 
                 7.33E−09 
                 Inhibitory 
               
               
                 1.62412777 
                 0.23611234 
                 6.87862308 
                 6.04E−12 
                 Arpc1b 
                 Adnp 
                 1.03E−07 
                 ODC 
               
               
                 1.01957062 
                 0.14969889 
                 6.81080958 
                 9.71E−12 
                 Hapln1 
                 Adnp 
                 1.64E−07 
                 Inhibitory 
               
               
                 1.56023689 
                 0.23271119 
                 6.7046062 
                 2.02E−11 
                 Arpc1b 
                 Ddx3x 
                 3.38E−07 
                 ODC 
               
               
                 −1.5108351 
                 0.22672881 
                 −6.663622 
                 2.67E−11 
                 Plp1 
                 Larp4b 
                 4.42E−07 
                 ODC 
               
               
                 −1.2393766 
                 0.18747519 
                 −6.6108833 
                 3.82E−11 
                 Cldn11 
                 Ash1l 
                 6.26E−07 
                 ODC 
               
               
                 1.56680536 
                 0.23826451 
                 6.57590736 
                 4.84E−11 
                 Ptgds 
                 Cul3 
                 7.85E−07 
                 Excitatory 
               
               
                 −0.6337373 
                 0.09655679 
                 −6.5633633 
                 5.26E−11 
                 Plp1 
                 Spen 
                 8.45E−07 
                 ODC 
               
               
                 3.33288756 
                 0.51125934 
                 6.51897632 
                 7.08E−11 
                 Hba-a2 
                 Setd2 
                 1.13E−06 
                 Excitatory 
               
               
                 1.46508737 
                 0.2268308 
                 6.45894359 
                 1.05E−10 
                 Arpc1b 
                 Wac 
                 1.66E−06 
                 ODC 
               
               
                 4.20640549 
                 0.6563858 
                 6.40843466 
                 1.47E−10 
                 Npy 
                 Mbd5 
                 2.29E−06 
                 Inhibitory 
               
               
                 2.28899941 
                 0.36598588 
                 6.25433801 
                 3.99E−10 
                 Ptgds 
                 Tcf20 
                 6.17E−06 
                 Inhibitory 
               
               
                 −0.883221 
                 0.14697577 
                 −6.0092966 
                 1.86E−09 
                 Plp1 
                 Chd8 
                 2.85E−05 
                 ODC 
               
               
                 −0.7019466 
                 0.11725546 
                 −5.986473 
                 2.14E−09 
                 Ntrk2 
                 Ctnnb1 
                 3.25E−05 
                 Astroglia 
               
               
                 4.61448205 
                 0.7768471 
                 5.94001325 
                 2.85E−09 
                 Ttr 
                 Tcf7l2 
                 4.28E−05 
                 Microglia 
               
               
                 0.73546474 
                 0.12416788 
                 5.92314805 
                 3.16E−09 
                 Hapln1 
                 Ctnnb1 
                 4.70E−05 
                 Inhibitory 
               
               
                 4.00977439 
                 0.67920703 
                 5.90361146 
                 3.56E−09 
                 Npy 
                 Setd5 
                 5.24E−05 
                 Inhibitory 
               
               
                 0.84683051 
                 0.14410136 
                 5.87663094 
                 4.19E−09 
                 Hapln1 
                 Myst4 
                 6.12E−05 
                 Inhibitory 
               
               
                 4.32983766 
                 0.74585461 
                 5.8052033 
                 6.43E−09 
                 Hbb-bs 
                 Wac 
                 9.30E−05 
                 Microglia 
               
               
                 −2.9513063 
                 0.51244643 
                 −5.7592484 
                 8.45E−09 
                 Cldn11 
                 Med13l 
                 0.00012119 
                 ODC 
               
               
                 −2.5460124 
                 0.45151862 
                 −5.638776 
                 1.71E−08 
                 Plp1 
                 Ank2 
                 0.00024349 
                 ODC 
               
               
                 −2.3640383 
                 0.42321597 
                 −5.5858911 
                 2.33E−08 
                 Cldn11 
                 Chd8 
                 0.00032483 
                 ODC 
               
               
                 −0.3327094 
                 0.05956259 
                 −5.5858786 
                 2.33E−08 
                 Mbp 
                 Scn2a1 
                 0.00032483 
                 ODC 
               
               
                 3.37933174 
                 0.61620308 
                 5.48412013 
                 4.16E−08 
                 Hbb-bt 
                 Setd2 
                 0.00057549 
                 Excitatory 
               
               
                 −0.6644442 
                 0.12248928 
                 −5.4245093 
                 5.81E−08 
                 Ntrk2 
                 Fbxo11 
                 0.00079797 
                 Astroglia 
               
               
                 5.75439332 
                 1.0622646 
                 5.41709975 
                 6.06E−08 
                 Hbb-bs 
                 Chd2 
                 0.00082469 
                 Microglia 
               
               
                 0.28337487 
                 0.0524902 
                 5.39862455 
                 6.72E−08 
                 Mbp 
                 Ctnnb1 
                 0.0009066 
                 ODC 
               
               
                 −0.6336752 
                 0.1182088 
                 −5.360643 
                 8.29E−08 
                 Ntrk2 
                 Stard9 
                 0.0011102 
                 Astroglia 
               
               
                 1.50091129 
                 0.28184137 
                 5.32537609 
                 1.01E−07 
                 Hbb-bs 
                 Mbd5 
                 0.0013376 
                 Excitatory 
               
               
                 −0.6875995 
                 0.13260917 
                 −5.1851583 
                 2.16E−07 
                 Ntrk2 
                 Scn2a1 
                 0.00284214 
                 Astroglia 
               
               
                 −0.4819589 
                 0.0936245 
                 −5.1477862 
                 2.64E−07 
                 Plp1 
                 Ash1l 
                 0.00341268 
                 ODC 
               
               
                 1.20218873 
                 0.23354092 
                 5.1476578 
                 2.64E−07 
                 Arpc1b 
                 Stard9 
                 0.00341268 
                 ODC 
               
               
                 0.61606747 
                 0.11970841 
                 5.1464011 
                 2.66E−07 
                 Cldn11 
                 Tnrc6b 
                 0.00341268 
                 ODC 
               
               
                 −0.6989003 
                 0.13756709 
                 −5.0804323 
                 3.77E−07 
                 Ntrk2 
                 Spen 
                 0.00480146 
                 Astroglia 
               
               
                 −0.7755879 
                 0.15313301 
                 −5.0647991 
                 4.09E−07 
                 Ntrk2 
                 Adnp 
                 0.00517167 
                 Astroglia 
               
               
                 −0.6123133 
                 0.12103527 
                 −5.0589657 
                 4.22E−07 
                 Ntrk2 
                 Wac 
                 0.00529073 
                 Astroglia 
               
               
                 1.38128024 
                 0.27525691 
                 5.01814926 
                 5.22E−07 
                 Arpc1b 
                 Gatad2b 
                 0.00649733 
                 ODC 
               
               
                 −0.7159794 
                 0.14276983 
                 −5.0149208 
                 5.31E−07 
                 Ntrk2 
                 Syngap1 
                 0.00655656 
                 Astroglia 
               
               
                 0.62367795 
                 0.12452019 
                 5.0086491 
                 5.48E−07 
                 Ndufs6 
                 Spen 
                 0.00672211 
                 Inhibitory 
               
               
                 −0.5816956 
                 0.11628509 
                 −5.0023233 
                 5.66E−07 
                 Ntrk2 
                 Tnrc6b 
                 0.00689392 
                 Astroglia 
               
               
                 3.36831358 
                 0.67386477 
                 4.99850081 
                 5.78E−07 
                 Npy 
                 Wac 
                 0.0069791 
                 Inhibitory 
               
               
                 −0.6435378 
                 0.12916958 
                 −4.9821154 
                 6.29E−07 
                 Ntrk2 
                 Ddx3x 
                 0.00754027 
                 Astroglia 
               
               
                 3.38642605 
                 0.68725869 
                 4.92744014 
                 8.33E−07 
                 Npy 
                 Ash1l 
                 0.00991457 
                 Inhibitory 
               
               
                 −1.0102543 
                 0.2075061 
                 −4.8685523 
                 1.12E−06 
                 Clu 
                 Adnp 
                 0.01327976 
                 Astroglia 
               
               
                 −0.6560352 
                 0.13537765 
                 −4.8459641 
                 1.26E−06 
                 Prnp 
                 Ddx3x 
                 0.01477523 
                 Astroglia 
               
               
                 −0.5893215 
                 0.12244623 
                 −4.8129005 
                 1.49E−06 
                 Ntrk2 
                 Upf3b 
                 0.01731745 
                 Astroglia 
               
               
                 −0.8949596 
                 0.18834467 
                 −4.7517119 
                 2.02E−06 
                 Clu 
                 Setd2 
                 0.02331228 
                 Astroglia 
               
               
                 3.56988215 
                 0.7515853 
                 4.74980305 
                 2.04E−06 
                 Ttr 
                 Fbxo11 
                 0.02336531 
                 Inhibitory 
               
               
                 −0.5087962 
                 0.10742482 
                 −4.7363005 
                 2.18E−06 
                 Mbp 
                 Tcf7l2 
                 0.0247635 
                 ODC 
               
               
                 2.96267387 
                 0.62567552 
                 4.73516031 
                 2.19E−06 
                 Npy 
                 Tnrc6b 
                 0.0247635 
                 Inhibitory 
               
               
                 1.08505114 
                 0.23096742 
                 4.69785359 
                 2.63E−06 
                 Arpc1b 
                 Tnrc6b 
                 0.02953663 
                 ODC 
               
               
                 2.16848391 
                 0.46218431 
                 4.69181638 
                 2.71E−06 
                 Hbb-bs 
                 Upf3b 
                 0.03021068 
                 Inhibitory 
               
               
                 −2.403628 
                 0.51248771 
                 −4.6901183 
                 2.73E−06 
                 Cldn11 
                 Larp4b 
                 0.03025239 
                 ODC 
               
               
                 −0.8675184 
                 0.18534916 
                 −4.6804548 
                 2.86E−06 
                 Plp1 
                 Pten 
                 0.0314968 
                 ODC 
               
               
                 −0.7529819 
                 0.16208886 
                 −4.6454887 
                 3.39E−06 
                 Mbp 
                 Satb2 
                 0.03707843 
                 ODC 
               
               
                 0.98248998 
                 0.21234995 
                 4.62674924 
                 3.71E−06 
                 Tsc22d1 
                 Spen 
                 0.04032075 
                 Inhibitory 
               
               
                 −0.4464993 
                 0.09766198 
                 −4.571884 
                 4.83E−06 
                 Ntrk2 
                 Mll1 
                 0.05211626 
                 Astroglia 
               
               
                 0.68408923 
                 0.14977381 
                 4.56748238 
                 4.94E−06 
                 Arpp19 
                 Setd5 
                 0.05286413 
                 Astroglia 
               
               
                 2.54395958 
                 0.5572094 
                 4.56553603 
                 4.98E−06 
                 Sox9 
                 Chd8 
                 0.05286413 
                 Inhibitory 
               
               
                 0.38752117 
                 0.08489488 
                 4.56471795 
                 5.00E−06 
                 Rpl13 
                 Adnp 
                 0.05286413 
                 Astroglia 
               
               
                 1.17339961 
                 0.25715884 
                 4.56293703 
                 5.04E−06 
                 Taldo1 
                 Setd5 
                 0.05296625 
                 Inhibitory 
               
               
                 0.93008445 
                 0.2045507 
                 4.54696286 
                 5.44E−06 
                 Hnrnpr 
                 Arid1b 
                 0.05677703 
                 Inhibitory 
               
               
                 2.23421275 
                 0.4915416 
                 4.54531769 
                 5.49E−06 
                 Gpr12 
                 Spen 
                 0.05685316 
                 Inhibitory 
               
               
                 2.16012919 
                 0.48047013 
                 4.4958657 
                 6.93E−06 
                 Zbtb20 
                 Ank2 
                 0.07080412 
                 Excitatory 
               
               
                 1.35202256 
                 0.30074561 
                 4.49556872 
                 6.94E−06 
                 Smc5 
                 Larp4b 
                 0.07080412 
                 Excitatory 
               
               
                 −0.5905943 
                 0.13139495 
                 −4.4948022 
                 6.96E−06 
                 Ntrk2 
                 Setd5 
                 0.07080412 
                 Astroglia 
               
               
                 0.99702063 
                 0.22241062 
                 4.48279229 
                 7.37E−06 
                 Mrps18c 
                 Arid1b 
                 0.07443878 
                 Inhibitory 
               
               
                 1.8859329 
                 0.42113384 
                 4.47822693 
                 7.53E−06 
                 C2cd4c 
                 Larp4b 
                 0.07557298 
                 Excitatory 
               
               
                 1.11874681 
                 0.2498978 
                 4.47681735 
                 7.58E−06 
                 Arpc1b 
                 Myst4 
                 0.07560096 
                 ODC 
               
               
                 −0.6055725 
                 0.1353468 
                 −4.4742282 
                 7.67E−06 
                 Ntrk2 
                 Setd2 
                 0.07605041 
                 Astroglia 
               
               
                 1.14072146 
                 0.25568367 
                 4.46145611 
                 8.14E−06 
                 Arpc1b 
                 Setd2 
                 0.08023274 
                 ODC 
               
               
                 0.64779745 
                 0.14527577 
                 4.45908805 
                 8.23E−06 
                 Hapln1 
                 Ash1l 
                 0.08062955 
                 Inhibitory 
               
               
                 0.88618226 
                 0.19892483 
                 4.45485989 
                 8.39E−06 
                 Arpc1b 
                 Dyrk1a 
                 0.08119682 
                 ODC 
               
               
                 1.08547175 
                 0.24370886 
                 4.45396924 
                 8.43E−06 
                 Tada1 
                 Arid1b 
                 0.08119682 
                 Excitatory 
               
               
                 0.86721561 
                 0.19471817 
                 4.45369638 
                 8.44E−06 
                 Dgcr6 
                 Arid1b 
                 0.08119682 
                 Astroglia 
               
               
                 −0.6822827 
                 0.15333088 
                 −4.4497408 
                 8.60E−06 
                 Lsm5 
                 Dscam 
                 0.08221442 
                 Excitatory 
               
               
                 0.36259888 
                 0.08158909 
                 4.4442078 
                 8.82E−06 
                 Rps10 
                 Tnrc6b 
                 0.08385952 
                 Astroglia 
               
               
                 3.46651448 
                 0.78048929 
                 4.44146323 
                 8.93E−06 
                 Hbb-bs 
                 Scn2a1 
                 0.08443685 
                 Microglia 
               
               
                 −0.4935346 
                 0.11130412 
                 −4.4341092 
                 9.25E−06 
                 Cpe 
                 Adnp 
                 0.08685975 
                 Astroglia 
               
               
                 1.1449619 
                 0.25933007 
                 4.41507578 
                 1.01E−05 
                 Sfpq 
                 Dscam 
                 0.09431352 
                 ODC 
               
               
                 1.06384831 
                 0.24135437 
                 4.40782707 
                 1.04E−05 
                 Taldo1 
                 Stard9 
                 0.09696122 
                 Inhibitory 
               
               
                 −0.6849412 
                 0.15552345 
                 −4.4041028 
                 1.06E−05 
                 Camk2g 
                 Mll1 
                 0.09807489 
                 Inhibitory 
               
               
                 1.11130918 
                 0.25354834 
                 4.3830268 
                 1.17E−05 
                 Txn2 
                 Gatad2b 
                 0.10697917 
                 Microglia 
               
               
                 1.70644766 
                 0.38935676 
                 4.38273543 
                 1.17E−05 
                 Zfp422 
                 Larp4b 
                 0.10697917 
                 Astroglia 
               
               
                 −0.518819 
                 0.11860238 
                 −4.3744401 
                 1.22E−05 
                 Ntrk2 
                 Asxl3 
                 0.11050104 
                 Astroglia 
               
               
                 2.33295455 
                 0.53349027 
                 4.37300303 
                 1.23E−05 
                 Cpsf3l 
                 Chd2 
                 0.11060634 
                 Astroglia 
               
               
                 −1.3170531 
                 0.30214946 
                 −4.3589458 
                 1.31E−05 
                 Leng1 
                 Dyrk1a 
                 0.11666096 
                 Astroglia 
               
               
                 3.14734913 
                 0.72204898 
                 4.35891359 
                 1.31E−05 
                 Tamm41 
                 Upf3b 
                 0.11666096 
                 Excitatory 
               
               
                 0.2506886 
                 0.05757107 
                 4.35441951 
                 1.33E−05 
                 mt-Co1 
                 Dscam 
                 0.11842204 
                 Excitatory 
               
               
                 0.30013747 
                 0.06901224 
                 4.34904685 
                 1.37E−05 
                 Rps10 
                 Mll1 
                 0.12069359 
                 Astroglia 
               
               
                 0.64521472 
                 0.148931 
                 4.33230649 
                 1.48E−05 
                 Agfg1 
                 Pogz 
                 0.12953706 
                 Excitatory 
               
               
                 −1.1307645 
                 0.26302777 
                 −4.299031 
                 1.72E−05 
                 Nnat 
                 Larp4b 
                 0.14925226 
                 Inhibitory 
               
               
                 −0.8278531 
                 0.19261385 
                 −4.2979936 
                 1.72E−05 
                 Cldn11 
                 Dscam 
                 0.14925226 
                 ODC 
               
               
                 1.77608614 
                 0.41329156 
                 4.29741695 
                 1.73E−05 
                 Polr3k 
                 Larp4b 
                 0.14925226 
                 Astroglia 
               
               
                 0.98747829 
                 0.23049792 
                 4.2841093 
                 1.83E−05 
                 Arpc1b 
                 Kdm5b 
                 0.15762317 
                 ODC 
               
               
                   
               
            
           
         
       
     
     GO Term Gene Signatures 
     The mm10 GO ontology was downloaded, and terms with &gt;500 or &lt;5 genes were removed. For each GO Term and each cell type, the genes in that term that appeared in &lt;5% of cells of that cell types were removed. For each term the average logTPM expression score was calculated and a linear regression model was fit to this score incorporating nGene, batch, and perturbation as covariates. P-values and effect sizes for each perturbation (relative to GFP) were calculated, and FDR correction was performed. 
     Analysis of Human Single Nucleus or Single Cell RNA-Seq Data 
     For each single cell/nucleus human dataset, the UMI count matrix and metadata were downloaded from their website (https://autism.cells.ucsc.edu/) and processed with Seurat to create Seurat objects. Cell types were extracted from the metadata and were combined into more general cell types, namely: Microglia, Astroglia (including Radial Glia), Inhibitory neurons, Excitatory neurons, Oligodendrocytes, and other. For differential expression analysis for data from Velmeshev et al (30), data from all individuals of &lt;12 years of age was removed and separated PFC and ACC regions. For each cell type in each region, a pseudobulk profile was constructed and genes expressed in &lt;5% of cells or with &lt;10 reads were removed. DESeq2 v 1.20.0 (34) was then used to perform differential expression analysis between the ASD patients and the controls, correcting for sex and age (note: age was encoded as a discrete value, not continuous). All genes were then extracted with 1:1 mouse orthologs (BioMart) and calculated FDR corrected P-values on these genes for both ACC and PFC. Only analysis on the PFC yielded significant hits, which are presented in  FIG. 4F . 
     To compare these results to the Perturb-Seq data, for each gene and cell type in the final DE table produced (data not shown), the median log fold change (log FC) for that gene&#39;s mouse orthologue over all perturbations was calculated from the Perturb-Seq data (see Differential expression analysis) and took the absolute value to get an absolute log FC score for each gene. Those genes for whom the sign (+1 or −1) of the median log FC agreed with the sign in the human data had their absolute log FC score multiplied by 1; those that disagreed had their absolute log FC score multiplied by −1, such that genes whose direction of change in the Perturb-Seq data agreed with the human data had positive scores, and those whose direction of change disagreed had negative scores. 
     Finally, genes were binned into 5% wide bins based on the % of cells expressing the gene in the Perturb-Seq data and assigned p-values to each gene based on the percent of genes in the same bin that had an equal or higher score. Finally, the list was filtered to contain only genes also differentially expressed in the human ASD data, and FDR correction was performed. 
     Scoring of PsychEncode Modules in Perturb-Seq Single Cell Data 
     PsychEncode modules were downloaded from the Science website, and 1:1 mouse orthologs were extracted for the genes in each module. The same linear regression analysis that was used on the WGCNA modules herein to determine effect size was applied to the PscyhEncode modules (using all cells instead of just one cell type), as was correlation analysis. 
     Cell Type Gene Expression 
     Expression data for the E18.5 mouse brain (9k dataset) was downloaded from the 10× website (10). The WT P7 data were generated from this paper. The P7 fastq files were run through the standard Cellranger pipeline. The data from both datasets were loaded into Seurat separately and transformed to log counts per million. Cells with &lt;500 genes were removed in both datasets. Variable genes were found using FindVariableGenes with x.low.cutoff=l, and the data was scaled with ScaleData, correcting for nUMI. PCA was performed, followed by TSNE and clustering with FindClusters. Cell types were identified with marker genes and contaminating/vascular cell types were removed. 
     In each dataset MAST (53) was used to find the differentially expressed genes in each cluster, relative to all cells outside that cluster. This was done correcting for the scaled nUMI and removing genes that occurred in less than 10 cells. Average expression was calculated for each gene in each cluster. 
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     Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.