TITLE: Generation of an isogenic set of HD model hESC lines
AUTHORS: Albert Ruzo, Gist F. Croft, Jakob J. Metzger, Szilvia Galgoczi, Lauren J. Gerber, Cecilia Pellegrini, Hanbin Wang Jr, Maria Fenner, Stephanie Tse, Adam Marks, Corbyn Nchako, Ali H. Brivanlou

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Huntington’s disease (HD) is a fatal neurodegenerative disease caused by expansion of CAG repeats in the Huntingtin gene (HTT). Neither its pathogenic mechanisms nor the normal functions of HTT are well understood. To model HD in humans, we engineered a genetic allelic series of isogenic human embryonic stem cell (hESC) lines with graded increases in CAG repeat length. Neural differentiation of these lines unveiled a novel developmental HD phenotype: the appearance of giant multinucleated telencephalic neurons at an abundance directly proportional to CAG repeat length, generated by a chromosomal instability and failed cytokinesis over multiple rounds of DNA replication. We conclude that disrupted neurogenesis during development is an important, unrecognized aspect of HD pathogenesis. To address the function of normal HTT protein we generated HTT+/- and HTT-/- lines. Surprisingly, the same phenotype emerged in HTT-/- but not HTT+/- lines. We conclude that HD is a developmental disorder characterized by chromosomal instability that impairs neurogenesis, and that HD represents a genetic dominant-negative loss of function, contrary to the prevalent gain-of-toxic-function hypothesis. The consequences of developmental alterations should be considered as a new target for HD therapies.</div></div>

[STEPS]
?. [Generation of an isogenic set of HD model hESC lines]
In order to establish an optimal platform for understanding the effects of HD mutations, use CRISPR/Cas9 technology to generate a set of isogenic hESC lines that would differ only in the HTT locus, bearing different lengths of the CAG tract.
?. [Generation of an isogenic set of HD model hESC lines]
Use the already established, registered and validated RUES2 hESC line (NIH hESC-09-0013) as the parental line.
Genetic analysis of the HTT gene in RUES2 showed that it contained 20 and 22 CAGs
?. [Generation of an isogenic set of HD model hESC lines]
Design the homology donors for the CRISPR/Cas9-mediated homologous recombination to contain ∼1 kb homology arms flanking HTT exon 1, a piggyBac transposable element containing a Puro-TK cassette (for both negative and positive selections), and various versions of HTT exon 1 containing different lengths of the CAG tract (20, 42, 48, 56, 67, 72 CAGs).
?. [Generation of an isogenic set of HD model hESC lines]
To construct the homology donors, first generate a parental plasmid using Gibson assembly, in which we could easily swap the length of the CAG tract.
?. [Generation of an isogenic set of HD model hESC lines]
Amplify all fragments needed for the assembly using Q5 high-fidelity polymerase (NEB) or the GC-RICH PCR System (SigmaAldrich) using the primers listed in Table S3.
This resulting ʻbase’ homology donor plasmid contained a 20 CAG repeat tract, which could easily be swapped using the flanking XmnI and BbsI sites.
?. [Generation of an isogenic set of HD model hESC lines]
To create the homology donor plasmids with expanded CAG lengths, amplify PCR the HTT exon 1 region from genomic DNAs obtained from iPSCs or fibroblasts derived from HD patients, and clone them into the ʻbase’ plasmid using XmnI and BbsI.
The primers used for the CAG tract amplification are polyCAG_Fw CCAAGATGGACGGCCGCTC andpolyCAG_Rv AGGACAAGGGAAGACCCAAG. The origin of the templates used for each CAG length are summarized in Table S4.
?. [Generation of an isogenic set of HD model hESC lines]
For the CRISPR/Cas9-mediated targeting, design multiple sgRNAs to recognize sequences near the CAG tract, but gene targeting attempts using a single sgRNA were unsuccessful.
However, an optimized strategy that combined two sgRNAs targeting sequences that flanked the CAG tract proved to be much more efficient in generating the desired homologous recombination: hHTT_sgRNA25 and hHTT_sgRNA14 (Table S5).
?. [Generation of an isogenic set of HD model hESC lines]
Clone these two sgRNAs into a Cas9 nickase expression vector (pX335 from the Feng Zhang lab, Addgene plasmid #42335).
?. [Generation of an isogenic set of HD model hESC lines]
Nucelofect both CRISPR plasmids, together with the appropriate homology donor into RUES2 cells using a Nucleofector II instrument and Cell Line Nucleofector Kit L (Lonza).
?. [Generation of an isogenic set of HD model hESC lines]
Add puromycin to the cultures 5 days after nucleofection, and keep in for 5 more days to ensure selection of correctly targeted clones.
?. [Generation of an isogenic set of HD model hESC lines]
Nucleofect puromycin-resistant cells with an excision-only piggyBac transposase (Transposagen), and a subsequent ganciclovir treatment selected clones in which the selection cassette had been correctly removed, leaving a minimal footprint (one extra nucleotide in the 5′UTR).
?. [Generation of an isogenic set of HD model hESC lines]
Pick and expand colonies derived from single cells for screening.
PCR amplification and Sanger sequencing identified correctly targeted clones, with no mutations in the CRISPR/Cas9 target sites or the piggyBac excision site. All positive clones were heterozygously targeted, which is a common finding in long genetic insertions via CRISPR/Cas9 mediated homologous recombination, and there appeared to be no preference as to which allele had been targeted.
?. [Generation of an isogenic set of HD model hESC lines]
Select two to three independent clones for each CAG length to serve as independent biological replicates.
?. [Generation of an isogenic set of HD model hESC lines]
Confirm normal HTT expression by western blot, and karyotype all lines by G-banding and aCGH analyses to assess their genomic integrity and CNV.
We found a variety of CNVs and loss of heterozygosity, within and outside of normal ranges (Fig. S2C, Table S2), but these did not correlate with any of the observed phenotypic differences and were not the result of gene editing per se, as they were also present in genetically unmodified clones.
?. [Generation of an isogenic set of HD model hESC lines]
Perform whole-genome sequencing at NY Genome Center (NYGC) using the TruSeq DNA PCR-Free Kit for Library Preparation and run in an Illumina HiSeqX sequencer.
?. [Generation of an isogenic set of HD model hESC lines]
Identify around 1000 unique single-nucleotide polymorphisms in each line, probably caused by a founder effect from a genetically variable parental RUES2 cell line.
?. [Generation of an isogenic set of HD model hESC lines]
Validate the pluripotency status and absence of differentiation of the clones through immunofluorescence staining of cells seeded and cultured on micropatterned coverslips (CYTOO).
?. [Generation of isogenic HTT+/− and HTT−/− hESC lines]
Use CRISPR/Cas9 technology to generate a set of isogenic hESC lines with different HTT gene dosages (HTT+/+, HTT+/-, HTT-/-).
?. [Generation of isogenic HTT+/− and HTT−/− hESC lines]
Use RUES2 hESC (NIH hESC-09-0013) as the parental line.
?. [Generation of isogenic HTT+/− and HTT−/− hESC lines]
To create HTT-/- and HTT+/- lines, use CRISPR/Cas9 to delete the first exon of HTT, ensuring that no protein fragment would be produced in the targeted (ʻnull’) allele.
An optimized strategy that combined two sgRNAs that flanked the first exon proved to be efficient in generating the desired deletion: hHTT_sgRNA22 and hHTT_sgRNA14 (Table S5).
?. [Generation of isogenic HTT+/− and HTT−/− hESC lines]
Clone these two sgRNAs into an SpCas9 expression vector (pX330 from the Feng Zhang lab), modify to coexpress a puromycin cassette to facilitate selection of transfected cells, thus maximizing the percentage of targeted colonies.
?. [Generation of isogenic HTT+/− and HTT−/− hESC lines]
Nucleofect both CRISPR plasmids into RUES2 cells using a Nucleofector II instrument and Cell Line Nucleofector Kit L (Lonza).
?. [Generation of isogenic HTT+/− and HTT−/− hESC lines]
Add puromycin to the cultures  after nucleofection, and keep in for a further  to select for transfected cells.
?. [Generation of isogenic HTT+/− and HTT−/− hESC lines]
Pick and expand colonies derived from the puromycin-resistant cells for screening.
PCR amplification and Sanger sequencing identified correctly targeted clones with the desired exon1 deletion. Two to three clones per genotype are identified and further validated as above.
?. [Neural differentiation]
Subject isogenic lines to a default neural induction protocol adapted from Shi et al. (2012).
?. [Neural differentiation]
In short, seed cultures in suspension, or in confluent adherent culture, at 0.5-1×106 cells/ml and feed every other day with N2B27 serum-free medium with ROCK inhibitor (, Abcam) for the first 2 days, TGFβ inhibition ( SB-431542, Sigma and  LDN-193189, StemGent) for the first 10 days, treated with  FGF8 (R&D Systems) from day 12-22 to maximize the frequency of CTIP2 (BCL11B)-positive cells, dissociate and seed on adherent substrate (polyornithine/laminin, Ibidi) at day 14, fix and analyze at day 19 or continue in culture with added BDNF (R&D Systems) and IGF1 (R&D Systems, ), cAMP (, R&D Systems) and ascorbic acid (, Sigma), until dissociation and reseeding on substrate at day 40, and fixation and analysis at day 45.
?. [Immunostaining and imaging]
Fix cultures with 4% paraformaldehyde (PFA), rinse twice with PBS.
?. [Immunostaining and imaging]
Permeabilize and quench cultures with wash buffer (PBS+0.1% Triton X-100) containing  glycine and 0.1% sodium azide, block (wash buffer, 10% normal donkey serum, 0.1% sodium azide) for , incubate with primary antibodies at  (for primary antibodies and dilutions, see Table S6).
4 °C
?. [Immunostaining and imaging]
Wash three times in wash buffer for  each, incubate with secondary antibodies (Alexa 488, 555, 594, 647, Molecular Probes; 1:500) and DAPI nuclear counterstain for  at, and then wash as above.
4 Room temperature
?. [Immunostaining and imaging]
For mitotic synchronization and microtubule visualization, synchronize cultures at G-to-M transition with nocadazole (; Abcam) for .
?. [Immunostaining and imaging]
Rinse and release for , fix for  at  in culture medium with 2% PFA, then incubate for  at  in microtubule stabilization buffer [0.1 M PIPES pH 6.9,  MgCl2,  EGTA, 2% formaldehyde, 0.1% Triton X-100,  paclitaxel (Sigma),  aprotinin (Sigma) and 50% deuterium oxide (Messinger and Albertini, 1991)] before quench and blocking as above.
4 Room temperature
37 °C
10
?. [Immunostaining and imaging]
Acquire Z-stack images on a Leica SP8 inverted confocal microscope at 12 bits in 1024 pixels ×1024 pixels using an HCX PL APO CS ×20/0.75 NA airimmersion objective, an HC PL APO CS2×40/1.10 NA water-immersion objective, or an HC PL APO ×100/1.4 oil-immersion objective (Leica) at Nyquist spacings.
?. [Immunostaining and imaging]
Deconvolve images with a 3D blind algorithm (ten iterations) using AutoDeblur X software (Autoquant) and rendered in Imaris (Bitplane).
?. [Immunostaining and imaging]
Subject 3D rendering channels to uniform non-linear adjustment across the entire volume to highlight salient details; adjustments are identical across genotypes.
?. [Analysis of neural rosette spatial distribution]
Identify rosettes by training a classifier on N-cadherin antibody stains using Ilastik (Sommer et al., 2011).
?. [Analysis of neural rosette spatial distribution]
Threshold N-cadherin-positive areas in size (15 µm2) to eliminate small N-cadherin spots that are not fully developed rosettes.
The quantification results are not dependent on the specific value chosen for the threshold.
?. [Analysis of neural rosette spatial distribution]
Obtain the nearest-neighbor distributions using the k-d tree implementation in scikit-learn (http://scikit-learn.org) and are compare with the random distribution using the one-sample Kolmogorov–Smirnov distance between them.
The cumulative nearest-neighbor distribution F for a random set of points in two dimensions can easily be calculated asF(d) = 1 – e –λπd2where λ is the 2D density of foci, and d is the distance between two neighbors. We rescaled the data for the individual lines separately according to their λ.
?. [Time-lapse imaging]
Dissociate neural cultures to single cells at day 25, and reseed at 0.5×106/cm2 on polyornithine laminin-coated 35 mm imaging dishes (Ibidi).
?. [Time-lapse imaging]
One day later, tranduce cultures with GFP and RFP nucleus-labeling baculoviruses (CellLight BacMam 2.0, ThermoFisher Scientific) for .
?. [Time-lapse imaging]
Two days later, when the nuclear signal was clearly observable, carry out a time-lapse imaging acquisition for  using a CellVoyager CV1000 spinning-disk confocal microscope (Yokogawa).