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FokI-dCas9 mediates high-fidelity genome editing in pigs.
Fisicaro, Nella; Salvaris, Evelyn J; Philip, Gayle K; Wakefield, Matthew J; Nottle, Mark B; Hawthorne, Wayne J; Cowan, Peter J.
Afiliación
  • Fisicaro N; Immunology Research Centre, St Vincent's Hospital Melbourne, Melbourne, Vic., Australia.
  • Salvaris EJ; Immunology Research Centre, St Vincent's Hospital Melbourne, Melbourne, Vic., Australia.
  • Philip GK; Melbourne Bioinformatics, University of Melbourne, Melbourne, Vic., Australia.
  • Wakefield MJ; Melbourne Bioinformatics, University of Melbourne, Melbourne, Vic., Australia.
  • Nottle MB; Walter and Eliza Hall Institute, Melbourne, Vic., Australia.
  • Hawthorne WJ; Robinson Research Institute & Adelaide Medical School, University of Adelaide, Adelaide, SA, Australia.
  • Cowan PJ; Department of Surgery, Westmead Clinical School, Westmead Hospital, University of Sydney, Sydney, NSW, Australia.
Xenotransplantation ; 27(1): e12551, 2020 01.
Article en En | MEDLINE | ID: mdl-31407391
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Desoxirribonucleasas de Localización Especificada Tipo II / Biología Computacional / Edición Génica / Proteína 9 Asociada a CRISPR / Mutación Límite: Animals Idioma: En Revista: Xenotransplantation Asunto de la revista: TRANSPLANTE Año: 2020 Tipo del documento: Article País de afiliación: Australia Pais de publicación: Dinamarca
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Desoxirribonucleasas de Localización Especificada Tipo II / Biología Computacional / Edición Génica / Proteína 9 Asociada a CRISPR / Mutación Límite: Animals Idioma: En Revista: Xenotransplantation Asunto de la revista: TRANSPLANTE Año: 2020 Tipo del documento: Article País de afiliación: Australia Pais de publicación: Dinamarca