FokI‐dCas9 mediates high‐fidelity genome editing in pigs

• Published in: Xenotransplantation (Københaven) 2020-01, Vol.27 (1), p.e12551-n/a
• Main Authors: Fisicaro, Nella, Salvaris, Evelyn J., Philip, Gayle K., Wakefield, Matthew J., Nottle, Mark B., Hawthorne, Wayne J., Cowan, Peter J.
• Format: Article
• Language: English
• Subjects: Animals; Chromosome 18; Chromosome 6; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Computational Biology - methods; Computer programs; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems; Deoxyribonucleases, Type II Site-Specific - genetics; Deoxyribonucleic acid; DNA; DNA Mutational Analysis; Feasibility Studies; Fidelity; Gene duplication; Gene Editing - methods; Genomes; knock‐in pig; Mutagenesis; Mutation; Mutation - genetics; off‐target event; Sus scrofa; Transplantation, Heterologous; Whole Genome Sequencing; Xenografts; xenotransplantation
• ISSN: 0908-665X; 1399-3089
• DOI: 10.1111/xen.12551

Gene editing using clustered regularly interspaced short palindromic repeats/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off‐target mutations in gene‐edited pigs remains largely undefined. We have previously generated GGTA1 knock‐in pigs for xenotransplantation using FokI‐dCas9, a variant of Cas9 that is reported to reduce the frequency of off‐target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of FokI‐dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using GRIDSS variant‐calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock‐in and wild‐type pigs. Platypus variant‐calling software was used to detect single‐nucleotide variations (SNVs) and small insertions/deletions (indels). GRIDSS analysis confirmed the precise integration of one copy of the knock‐in construct in the gene‐edited cells. Three additional SVs were detected by GRIDSS: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the CALD1 gene on chromosome 18. These mutations were not associated with plausible off‐target sites, and were not detected in a second line of knock‐in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off‐target activity. Platypus identified 1375 SNVs/indels after quality filtering, but none of these were located in proximity to potential off‐target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from FokI‐dCas9‐edited cells. Our results demonstrate that FokI‐dCas9 is capable of high‐fidelity gene editing with negligible off‐target or undesired on‐target mutagenesis.