Developed enhanced-specificity Cas9 (eSpCas9) through charge neutralization in non-target strand binding groove. Reduced off-targets while preserving on-target activity. Complementary approach to HF1.
Rationally engineered Cas9 nucleases with improved specificity
Cas9 cleaves off-target sites partially complementary to its guide RNA. Structure-guided engineering produced 'enhanced specificity' eSpCas9, validated by genome-wide sequencing. eSpCas9 cuts off-target sites less, keeping on-target activity near wild-type.
Parts used
Key findings
Abstract
The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we 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, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.