Once the RNA-guided endonuclease Cas9 from microbial clustered regularly-interspaced short palindromic repeat (CRISPR)/Cas adaptive immune systems was developed––as a powerful tool for genome editing in eukaryotic cells––everyone knew it was only a matter of time until even that breakthrough could be imporved upon.
CRISPR/Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to plant genetic engineering to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci that are complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a challenge for correct genome editing only. In the attached article, authors use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, they demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants diminish off-target effects, while maintaining robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.
Science 1 Jan 2o16; 351: 84–88