DOI: 10.5061/dryad.tqjq2bwbj
title: Data from: Engineered nucleocytosolic vehicles for loading of programmable editors

description: Advanced gene editing methods have accelerated biomedical discovery and
 hold great therapeutic promise, but safe and efficient delivery of gene
 editors remains challenging. In this study, we present a virus-like
 particle (VLP) system featuring nucleocytosolic shuttling vehicles that
 retrieve pre-assembled Cas-effectors via aptamer-tagged guide RNAs. This
 approach ensures preferential loading of fully assembled editor
 ribonucleoproteins (RNPs) and enhances the efficacy of prime editing, base
 editing, trans-activators, and nuclease activity coupled to
 homology-directed repair in multiple immortalized, primary, stem cell, and
 stem-cell-derived cell types. We also achieve additional protection of
 inherently unstable prime editing guide RNAs (pegRNAs) by shielding the
 3'-exposed end with Csy4/Cas6f, further enhancing editing
 performance. Furthermore, we identify a minimal set of packaging and
 budding modules that can serve as a platform for bottom-up engineering of
 enveloped delivery vehicles. Notably, our system demonstrates superior
 per-VLP editing efficiency in primary T lymphocytes and two mouse models
 of inherited retinal disease, highlighting its therapeutic potential.
description: # Data from: Engineered nucleocytosolic vehicles for loading of
 programmable editors \[10.1016/j.cell.2025.03.015] Data collection for raw
 sequencing data, raw imaging files, and other data associated with the
 corresponding publication. ## Description of the data and file structure *
 "2Photon_in_vivo_imaging.zip" contains the raw data of retinal
 images associated with the results presented in figure 7 and S14 in the
 manuscript. There are subfolders for the raw images generated for each eye
 explored in the manuscript, the folders are divided into ENVLPE and
 subsequent include every treated eye with extensive imaging from the
 respective area of the retina, both in black and white and additional the
 colored images. "ENVLP.zip" file group contains the images of
 subject F (female), NP (no punch, mouse identification), injected with
 1.5uL in the right eye (OD) with individual colored and black and white
 image reconstruction of the eye. Each of the individual images extracted
 from portions of the mouse eye contain scales in the left corner for
 consideration. Related information can be found in the published
 manuscript. "Fig S14 Fem OS 2uL.zip" file group contains the
 data for Figure S14 of female (F), 1R1L (mouse identification) injected
 with 2uL of ENVLPE in the left eye (OS). "Fig S14,1uL, Male
 OS.zip" describes the data of corresponding mouse male (M),
 identified with 1R punch (mouse identification) injected with 1uL of
 ENVLPE in the right eye (OD). "Fig S14, Fem OD 2uL.zip" features
 the subfolder Fem 2uL OD, which includes the 3-dimension reconstruction of
 images generated in Figures S14 of female (F), injected with 2uL of ENVLPE
 in the left eye (OS). "Figure S14 - 1uL OD.zip" features the
 data of male mice (M), identified with 1R punch (mouse identification),
 injected with 1uL of ENVLPE into the right eye (OD). "Male OD 1.5 UL
 Fig S14.zip" contains the raw data files of male mice (M), identified
 with punch 2R (2R), injected with 1.5uL of ENVLPE (1.5uL) into the right
 eye (OD). * "ERG_in_vivo_rd12.zip" contains the analyzed
 electroretinography (ERG) data for each individual rd12 mouse analyzed
 included in Figure 7 in the manuscript. Each Overview tab includes the
 individual tag number per each mouse treated with the indicated injection
 date listed, the treatment received in each eye and the cage pertaining
 that each mouse included pertained to. The best responding mouse out of
 each treatment groups (ENVLPE and eVLP) have individual tabs including all
 the raw ERG traces recorded and the tabs are listed according to the mouse
 tag number and OS/OD assignements pertaining to left eye (OS) or right eye
 (OD) in which the best response out of each treatment was included. The
 Overview CTRL tab describes the listed non-treated rd12 mouse with cage
 number and individual name assigned and the - PBS control tab and utx tab
 correspond to the buffer control treated mice and untreated mice,
 respectively. The WT1 tab includes the rata data files of individual
 wildtype mice recorded for representative trace included in Figure 7. The
 data is divided into a and B waves for each individual mouse recording
 that was integrated into Figure 7. The subtabs divide the treatment group
 arms into eVLP and ENVLPE treatments and there are subtabs including the
 raw data from the best responding rd12 animal from each of the treated
 groups. Related information can be found in the published manuscript.
 "Full experiment" tab includes the treatment group
 identification in row 1: "ENVLPE RPE65"" or "eVLP
 RPE65" with the corresponding cage number for each group listed in
 columns A and M and the date of birth (DOB) in columns B, F and S. An
 individual name tag is associated to each mouse, listed by cage in columns
 C and O and the individual treatment for each mouse in columns G and H for
 ENVLPE and columns T and U for eVLP-treated mice. "Overview - ENVLPE
 tab"" includes the duplicate columns extracted from the
 "Full experiment" tab to list only the mice treated with ENVLPE
 and the "ENVLPE" tab includes the raw data results for each
 individual mouse with corresponding a wave values for right eye (OD a) and
 left eye (OS a) and b wave values for right eye (OD B) and left eye (OS
 B). The same exact parameters are repeated for eVLP including the full
 list of animals extracted from "Full experiment tab" in
 "Overview - eVLP" and the raw data values and a and B data in
 "eVLP RPE65 1.13". Further detail can be found in the
 manuscript. * "Tissue_culture_quantification.zip" contains the
 detailed cell culture analysis and images associated with the results
 presented in the Figures 1-16. Each individual subtab of the file (named
 "mmc2") contains the quantification for each of the figures
 published in the manuscript, expanding from the individual raw datapoints
 included into each graph and the corresponding analysis for each of the
 comparison and results published in the manuscript.Each figure featured in
 the manuscript is further divided into panels that are separated into
 it's own tab composing all the raw data further published into the
 manuscript with the corresponding naming indicated in the tab, exemplified
 by tab 1A referring to Figure 1 of the manuscript and panel A that is also
 correspondingly listed as panel A in the manuscript. *
 "NGS_for_in_vivo_editing.zip" contains the raw data of NGS
 sequencing for rd12 editing and rd6 editing (Figure 7) in the manuscript.
 The rd12 files encompass the gene editing in the RPE65 locus whereas the
 rd6 are amplifying the editing in the MFRP locus. There are subfolders for
 the raw sequencing data for each individual mouse analyzed and the sample
 information file (named "Sample Information") lists each
 individual treatment corresponding the raw data subfolder. The
 "ENVLPE Sequencing"" file lists individual sequencing
 results data corresponding to each mouse treated with either ENVLPE or
 eVLP treatment arms and non-treated controls and wildtype values for
 references. Rd12 and rd6 editing in the RPE and retina of the
 corresponding rd12 and rd6 mouse models. *
 "VLP_transfection_components.zip" contains file (named
 "mmc1") the plasmid ratios utilized for each virus-like particle
 (VLP) formulation published throughout the manuscript, including eVLPs,
 ENVLPEs and mini-ENVLPEs, pegRNA sequences, protein and mRNA cargo
 packaged in each particle variant, primers utilized and formulations.
 Related information can be found in the published manuscript. *
 "Plasmid_maps.zip" contains the plasmid maps used in this study,
 encompassing the subcomponents of all the nucleocytosolic virus-like
 particle variants. Related information can be found in the published
 manuscript.