A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells
Web of Science
AuthorHowden, SE; McColl, B; Glaser, A; Vadolas, J; Petrou, S; Little, MH; Elefanty, AG; Stanley, EG
Source TitleStem Cell Reports
University of Melbourne Author/sPetrou, Steven; Little, Melissa; Glaser, Astrid; Howden, Sara; Vadolas, Jim; Elefanty, Andrew; Stanley, Edouard
AffiliationFlorey Department of Neuroscience and Mental Health
Document TypeJournal Article
CitationsHowden, S. E., McColl, B., Glaser, A., Vadolas, J., Petrou, S., Little, M. H., Elefanty, A. G. & Stanley, E. G. (2016). A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells. STEM CELL REPORTS, 7 (3), pp.508-517. https://doi.org/10.1016/j.stemcr.2016.07.001.
Access StatusOpen Access
While Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines, the CRISPR-Cas9 system can introduce undesirable "on-target" mutations within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address this, we fused the Streptococcus pyogenes Cas9 (SpCas9) nuclease to a peptide derived from the human Geminin protein (SpCas9-Gem) to facilitate its degradation during the G1 phase of the cell cycle, when DNA repair by NHEJ predominates. We also use mRNA transfection to facilitate low and transient expression of modified and unmodified versions of Cas9. Although the frequency of homologous recombination was similar for SpCas9-Gem and SpCas9, we observed a marked reduction in the capacity for SpCas9-Gem to induce NHEJ-mediated indels at the target locus. Moreover, in contrast to native SpCas9, we demonstrate that transient SpCas9-Gem expression enables reliable generation of both knockin reporter cell lines and genetically repaired patient-specific induced pluripotent stem cell lines free of unwanted mutations at the targeted locus.
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