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J Vis Exp. 2019 Feb 26;(144). doi: 10.3791/59202.

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution.

Author information

1
ToolGen.
2
ToolGen; jungjoon.lee@gmail.com.

Abstract

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance of its potentially deleterious off-target effects. Several methods have been devised to reduce such effects. Here, we present an Escherichia coli-based directed evolution method called Sniper-screen to obtain a Cas9 variant with optimized specificity and retained on-target activity, called Sniper-Cas9. Using Sniper-screen, positive and negative selection can be performed simultaneously. The screen can also be repeated with other single-guide RNA (sgRNA) sequences to enrich for the true positive hits. By using the CMV-PltetO1 dual promoter to express Cas9 variants, the performance of the pooled library can be quickly checked in mammalian cells. Methods to increase the specificity of Sniper-Cas9 are also described. First, the use of truncated sgRNAs has previously been shown to increase Cas9 specificity. Unlike other engineered Cas9s, Sniper-Cas9 retains a wild-type (WT) level of on-target activity when combined with truncated sgRNAs. Second, the delivery of Sniper-Cas9 in a ribonucleoprotein (RNP) format instead of a plasmid format is possible without affecting its on-target activity.

PMID:
30882797
DOI:
10.3791/59202
[Indexed for MEDLINE]

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