Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.
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.
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
Florian T
FT
Merkle
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA; Boston IVF, Waltham, MA 02451, USA.
Werner M
WM
Neuhausser
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
David
D
Santos
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; Computational Biology Unit, Department of Informatics, University of Bergen, 5020 Bergen, Norway.
Eivind
E
Valen
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
James A
JA
Gagnon
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA; Boston IVF, Waltham, MA 02451, USA.
Kristi
K
Maas
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Jackson
J
Sandoe
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Center for Brain Science, Harvard University, Cambridge, MA 02138, USA.
Alexander F
AF
Schier
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address: eggan@mcb.harvard.edu.
Kevin
K
Eggan
D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002999:Clone Cells; D055879:Gene Knock-In Techniques / Q000379:methods*; D018390:Gene Targeting; D056426:Genetic Loci; D006801:Humans; D054643:INDEL Mutation / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D039904:Pluripotent Stem Cells / Q000378:metabolism*