RRC ID 63004
Author Yokouchi Y, Suzuki S, Ohtsuki N, Yamamoto K, Noguchi S, Soejima Y, Goto M, Ishioka K, Nakamura I, Suzuki S, Takenoshita S, Era T.
Title Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing.
Journal Sci Rep
Abstract Many human diseases ranging from cancer to hereditary disorders are caused by single-nucleotide mutations in critical genes. Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases. However, current procedures for repairing deleterious single-nucleotide mutations are not straightforward, requiring multiple steps and taking several months to complete. In the current study, we aimed to repair pathogenic allele-specific single-nucleotide mutations using a single round of genome editing. Using high-fidelity, site-specific nuclease AsCas12a/Cpf1, we attempted to repair pathogenic single-nucleotide variants (SNVs) in disease-specific induced pluripotent stem cells. As a result, we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single-nucleotide marker, followed by absolute markerless, scarless repair of the RET SNV with no detected off-target effects. The markerless method was then confirmed in human type VII collagen-encoding gene COL7A1. Thus, using this One-SHOT method, we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one, resulting in allele-specific repair that can be completed within 3 weeks, with or without a single-nucleotide marker. Our findings suggest that One-SHOT can be used to repair other types of mutations, with potential beyond human medicine.
Volume 10(1)
Pages 13927
Published 2020-8-18
DOI 10.1038/s41598-020-70401-7
PII 10.1038/s41598-020-70401-7
PMID 32811847
PMC PMC7435196
MeSH Alleles Bacterial Proteins / genetics Bacterial Proteins / metabolism* CRISPR-Associated Proteins / genetics CRISPR-Associated Proteins / metabolism* CRISPR-Cas Systems / genetics Collagen Type VII / genetics Collagen Type VII / metabolism Endodeoxyribonucleases / genetics Endodeoxyribonucleases / metabolism* Endonucleases / genetics Gene Editing / methods* Genome, Human / genetics Humans Induced Pluripotent Stem Cells / physiology Mutation / genetics Nucleotides / genetics Pluripotent Stem Cells / physiology Polymorphism, Single Nucleotide / genetics* Proto-Oncogene Proteins c-ret / genetics Proto-Oncogene Proteins c-ret / metabolism
IF 3.998
Human and Animal Cells 409B2(HPS0076)