RRC ID 69780
Author Nakano Y, Susa K, Yanagi T, Hiraoka Y, Suzuki T, Mori T, Ando F, Mandai S, Fujiki T, Rai T, Uchida S, Sohara E.
Title Generation of NPHP1 knockout human pluripotent stem cells by a practical biallelic gene deletion strategy using CRISPR/Cas9 and ssODN.
Journal In Vitro Cell Dev Biol Anim
Abstract CRISPR/Cas9 genome editing underwent remarkable progress and significantly contributed to the development of life sciences. Induced pluripotent stem cells (iPSCs) have also made a relevant contribution to regenerative medicine, pharmacological research, and genetic disease analysis. However, knockout iPSC generation with CRISPR/Cas9 in general has been difficult to achieve using approaches such as frameshift mutations to reproduce genetic diseases with full-length or nearly full-length gene deletions. Moreover, splicing and illegitimate translation could make complete knockouts difficult. Full-length gene deletion methods in iPSCs might solve these problems, although no such approach has been reported yet. In this study, we present a practical two-step gene-editing strategy leading to the precise, biallelic, and complete deletion of the full-length NPHP1 gene in iPSCs, which is the first report of biallelic (compound heterozygous) full-gene deletion in iPSCs using CRISPR/Cas9 and single-stranded oligodeoxynucleotides mainly via single-strand template repair (SSTR). Our strategy requires no selection or substances to enhance SSTR and can be used for the analysis of genetic disorders that are difficult to reproduce by conventional knockout methods.
Volume 58(2)
Pages 85-95
Published 2022-2-1
DOI 10.1007/s11626-022-00655-0
PII 10.1007/s11626-022-00655-0
PMID 35165826
MeSH Adaptor Proteins, Signal Transducing / genetics Animals CRISPR-Cas Systems* / genetics Cytoskeletal Proteins / genetics Gene Deletion Gene Editing / methods Heterozygote Humans Induced Pluripotent Stem Cells*
IF 1.665
Human and Animal Cells 1231A3(HPS0381)