title: A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells.
abstract: 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.
authors: Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC 3052, Australia. Electronic address: sara.howden@mcri.edu.au.
authors: Sara E
authors: SE
authors: Howden
authors: Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia.
authors: Bradley
authors: B
authors: McColl
authors: Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC 3052, Australia.
authors: Astrid
authors: A
authors: Glaser
authors: Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC 3052, Australia.
authors: Jim
authors: J
authors: Vadolas
authors: The Florey Institute for Neuroscience and Mental Health, University of Melbourne, Parkville, VIC 3052, Australia.
authors: Steven
authors: S
authors: Petrou
authors: Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC 3052, Australia.
authors: Melissa H
authors: MH
authors: Little
authors: Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC 3052, Australia; Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC 3800, Australia.
authors: Andrew G
authors: AG
authors: Elefanty
authors: Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC 3052, Australia; Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC 3800, Australia.
authors: Edouard G
authors: EG
authors: Stanley
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D065150:Cellular Reprogramming / Q000235:genetics; D059766:DNA End-Joining Repair; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D064248:Geminin / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D055879:Gene Knock-In Techniques; D017930:Genes, Reporter; D006801:Humans; D054643:INDEL Mutation; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D052416:Targeted Gene Repair; D014162:Transfection
chemical_list: D064248:Geminin