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Cell Rep DOI:10.1016/j.celrep.2015.04.007

Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.

Publication TypeJournal Article
Year of Publication2015
AuthorsMerkle, FT, Neuhausser, WM, Santos, D, Valen, E, Gagnon, JA, Maas, K, Sandoe, J, Schier, AF, Eggan, K
JournalCell Rep
Date Published2015 May 12
KeywordsBase Sequence, Clone Cells, CRISPR-Cas Systems, Gene Knock-In Techniques, Gene Targeting, Genetic Loci, Humans, INDEL Mutation, Molecular Sequence Data, Mutation, Pluripotent Stem Cells

The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.


Alternate JournalCell Rep
PubMed ID25937281
PubMed Central IDPMC5533178
Grant List1R21NS071598 / NS / NINDS NIH HHS / United States
P01 GM099117 / GM / NIGMS NIH HHS / United States
5K99NS083713 / NS / NINDS NIH HHS / United States
HL109525 / HL / NHLBI NIH HHS / United States
/ / Howard Hughes Medical Institute / United States