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

Garmen Yuen, Fehad J. Khan, Shaojian Gao, Jayne M. Stommel, Eric Batchelor, Xiaolin Wu, Ji Luo

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)12039-12053
Number of pages15
JournalNucleic acids research
Volume45
Issue number20
DOIs
StatePublished - Nov 16 2017
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

Fingerprint

Dive into the research topics of 'CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level'. Together they form a unique fingerprint.

Cite this