DOI: 10.5061/dryad.rr4xgxdfh
title: Data from: Lung and liver editing using lipid nanoparticle delivery of a stable CRISPR-Cas9 RNP

description: Lipid nanoparticle (LNP) delivery of CRISPR ribonucleoproteins (RNPs)
 could enable high-efficiency, low-toxicity, and scalable in vivo genome
 editing if efficacious RNP:LNP complexes can be reliably produced. Here,
 we engineered a thermostable Cas9 from Geobacillus stearothermophilus
 (GeoCas9) to generate iGeoCas9 variants capable of &gt;100X more
 genome editing of cells and organs compared to the native GeoCas9 enzyme.
 Furthermore, iGeoCas9 RNP:LNP complexes edited a variety of cell types and
 induced homology-directed repair (HDR) in cells receiving co-delivered
 single-stranded DNA (ssDNA) templates. Using tissue-selective LNP
 formulations, we observed genome editing levels of 16‒37% efficiency in
 the liver and lungs of model reporter mice that received single
 intravenous injections of iGeoCas9 RNP:LNPs. In addition, iGeoCas9 RNP
 complexed to biodegradable LNPs edited the disease-causing SFTPC gene in
 lung tissue with 19% average efficiency, representing a major improvement
 over genome editing levels observed previously using viral or non-viral
 delivery strategies. These results show that thermostable Cas9 RNP:LNP
 complexes are a powerful alternative to mRNA:LNP delivery vehicles and
 expand the therapeutic potential of genome editing.
description: # Data from: Lung and liver editing using lipid nanoparticle delivery of a
 stable CRISPR-Cas9 RNP
 [https://doi.org/10.5061/dryad.rr4xgxdfh](https://doi.org/10.5061/dryad.rr4xgxdfh) Data collection for raw sequencing data, raw imaging files, and other data associated with the corresponding publication. ## Description of the data and file structure * "Geo-AAVS1-EMX1-CFTR-reorganized_raw_data" contains the raw data of NGS sequencing for Figure 4 and the related extended figures in the manuscript. There are subfolders for the raw sequencing data for each genomic site explored in the manuscript, including four AAVS1 sites (AAVS1-1 to AAVS1-4), four EMX1 sites (EMX1-1 to EMX1-4), and two examples of the CFTR gene (CFTR-542 and CFTR-1282). Related information can be found in the published manuscript. * "Final_NGS_analysis" contains the analyzed sequencing data for Figure 4 and the related extended figures in the manuscript. There are subfolders for the NGS analysis results for each genomic site explored in the manuscript, including four AAVS1 sites (AAVS1-1 to AAVS1-4), four EMX1 sites (EMX1-1 to EMX1-4), and two examples of the CFTR gene (CFTR-542 and CFTR-1282). Related information can be found in the published manuscript. * "Cell_culture_images" contains the cell culture images associated with the results presented in the supplementary materials. These files include images associated with EGFP-to-BFP editing and tdTom NPC editing described in our manuscript. * "Tissue_images" contains the liver or lung tissue images associated with Figures 6e and 6f in the manuscript. These files include images associated with genome editing experiments with Ai9 tdTom mouse models. Related information can be found in the published manuscript. * "NGS_for_in_vivo_PCSK9_editing" contains the raw data of NGS sequencing for PCSK9 editing (Figure 6h) in the manuscript. There are subfolders for the raw sequencing data for PCSK9 editing in the liver and the lungs of wild-type mouse models. * "NGS_for_in_vivo_SFTPC_editing" contains the raw data of NGS sequencing for SFTPC editing (Figure 6i) in the manuscript. There are subfolders for the raw sequencing data for SFTPC editing in the liver and the lungs of wild-type mouse models. * "NGS_for_off-target_analysis" contains the raw data of NGS sequencing for off-target effect analysis in the supplementary materials. There are subfolders for the raw sequencing data for each genomic site, on-target (named "XXX_ON") or off-target (named "XXX_OFT"). Folders named "Editing_XXX" contain the sequencing results from the group of genome editing experiments, and folders named "Neg Ctrl_XXX" contain the sequencing results from the group of negative controls. Detailed information on the on-/off-target sequences can be found in the manuscript. * "Plasmid_maps" contains the plasmid maps used in this study, including the evolutionary lineage of GeoCas9 variants. Related information can be found in the published manuscript.