Target specificity of the CRISPR-Cas9 system

Abstract

The CRISPR-Cas9 system, naturally a defense mechanism in prokaryotes, has been repurposed as an RNA-guided DNA targeting platform. It has been widely used for genome editing and transcriptome modulation, and has shown great promise in correcting mutations in human genetic diseases. Off-target effects are a critical issue for all of these applications. Here we review the current status on the target specificity of the CRISPR-Cas9 system.

Keywords: CRISPR; Cas9; genome engineering; off-targets; target specificity.

Figure 1. The CRISPR-Cas9 system

The sgRNA (purple) targets the Cas9 protein to genomic sites containing sequences complementary to the 5′ end of the sgRNA. The target DNA sequence needs to be followed by a proto-spacer adjacent motif (PAM), typically NGG. Cas9 is a DNA endonuclease with two active domains (red triangles) cleaving each of the two DNA strands three nucleotides upstream of the PAM. The five nucleotides upstream of the PAM are defined as the seed region for target recognition.

Figure 2. Factors that impact Cas9 specificity

(Top) Before Cas9 is introduced to the system, specificity can be modified by altering the architecture of the single guide RNA (sgRNA) or the Cas9 protein itself. (Middle) At the DNA level, beyond the PAM requirement for binding, closed chromatin and methylated DNA negatively impact Cas9 binding, while increased abundance of Cas9/sgRNA complexes and guide sequences in the genome positively impact Cas9 binding. (Bottom) Although Cas9 can transiently bind DNA that is complementary to only a small seed sequence in the sgRNA, only sequences with extensive complementarity to the guide will be cleaved or direct activation or silencing of targeted genes.