High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects

Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK
Source: Nature
Publication Date: (2016)
Issue: 529 (7587): 490-5
Research Area:
Gene Expression
Cells used in publication:
U-2 OS
Species: human
Tissue Origin: bone
Culture Media:
4D-Nucleofector® X-Unit
Unless otherwise noted, cells were co-transfected with 750 ng of Cas9 plasmid and 250 ng of sgRNA plasmid. For negative control experiments, Cas9 plasmids were co-transfected with a U6-null plasmid. Nucleofections were performed using the DN-100 program on a Lonza 4-D Nucleofector with the SE Cell Line Kit according to the manufacturer’s protocol (Lonza).
CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.