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Review
. 2015 Dec:29:72-8.
doi: 10.1016/j.cbpa.2015.10.001. Epub 2015 Oct 24.

How specific is CRISPR/Cas9 really?

Henriette O'Geen  1 , Abigail S Yu  1 , David J Segal  2
Affiliations
Review

How specific is CRISPR/Cas9 really?

Henriette O'Geen et al. Curr Opin Chem Biol. 2015 Dec.

Abstract

The specificity of RNA-guided nucleases has gathered considerable interest as they become broadly applied to basic research and therapeutic development. Reports of the simple generation of animal models and genome engineering of cells raised questions about targeting precision. Conflicting early reports led the field to believe that CRISPR/Cas9 system was promiscuous, leading to a variety of strategies for improving specificity and increasingly sensitive methods to detect off-target events. However, other studies have suggested that CRISPR/Cas9 is a highly specific genome-editing tool. This review will focus on deciphering and interpreting these seemingly opposing claims.

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Figures

Figure 1
Figure 1. CRISPR/Cas9 specificity is dependent on its sgRNA
The Cas9 protein (pink) complexes with a single guide RNA (sgRNA, red) at a DNA target site that contains a protospacer adjacent motif (PAM, blue box). The 5’ end of the sgRNA forms a 20-bp heteroduplex with one strand of the DNA. The binding or cleavage events facilitated by the Cas9/sgRNA complex can be categorized as highly specific (no off-targets), intermediate (1 to 5 off-targets) and promiscuous (6 or more off-targets).
Figure 2
Figure 2. A diversity of methods has been used to predict or detect off-target sites
Each successive methodology attempted to be less biased and interrogate more of the genome than the previous generation.
Figure 3
Figure 3. Some sgRNAs allow binding or cleavage at variants of the target site
In addition to single-base mismatches (a), some sgRNAs can tolerate DNA sequences with an extra base (b, DNA bulge) or a missing base (c, sgRNA bulge).
See this image and copyright information in PMC

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