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Nucleic Acids Res. 2017 Nov 16;45(20):12039-12053. doi: 10.1093/nar/gkx843.

CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

Author information

1
Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
2
Undergraduate Scholarship Program, National Institutes of Health, Bethesda, MD, USA.
3
Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
4
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
5
Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
6
Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA.

Abstract

CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform.

PMID:
29036671
PMCID:
PMC5714203
DOI:
10.1093/nar/gkx843
[Indexed for MEDLINE]
Free PMC Article

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