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Broad Scientists Engineer CRISPR/Cas9 Variants to Reduce Off-Target Effects, Maintain Efficiency

NEW YORK (GenomeWeb) – A new class of mutant Cas9 proteins can drastically reduce off-target activity while maintaining editing efficiency, according to a new report published today in Science.

Scientists from the Broad Institute led by co-first authors Ian Slaymaker and Linyi Gao and senior author Feng Zhang systematically interrogated a bevy of Cas9 mutants to find ones that improved genome editing specificity, an important consideration for clinical use of CRISPR/Cas9 genome editing. They identified three promising Cas9 mutants that showed wild-type levels of on-target activity at two loci with no detectable off-target effects. They have named this class of enzymes enhanced Streptococcus pyogenes Cas9s, or eSpCas9s.

"Our study suggests a generalizable strategy for enhancing the DNA targeting specificity of RNA-guided endonucleases," Slaymaker told GenomeWeb.

There have been several different existing strategies to decrease off-target effects of the Cas9 enzyme, including shortening the section of the guide RNA complementary to the target, deploying pairs of engineered nicking Cas9s, and reducing the amount of enzyme active in the cell; however, they all present certain limitations, often decreasing on-target editing efficiency.

The Broad researchers hypothesized that they could reduce the ability of Cas9 to bind with off-target sites by engineering the protein. They targeted a positively charged groove that interacts with the non-target strand of DNA, expecting that neutralizing it would "weaken non-target strand binding and encourage re-hybridization between the target and non-target strands, thereby requiring more stringent Watson-Crick base pairing between the RNA guide and the target DNA strand," the authors wrote.

They designed 32 mutant Cas9s, with an alanine substitution at 32 positively charged residues within the non-target strand groove. Five of these mutant Cas9s reduced off-target activity by at least ten-fold compared to wild type spCas9. The team then looked for combinations of mutations that would further reduce off-target effects: eight of the 35 mutants tested edited at wild-type efficiencies with no detectable off-target effects. The final three — SpCas9 (K855a), SpCas9 (K810A/K1003A/r1060A) or eSpCas9 1.0, and SpCas9 (K848A/K1003A/R1060A) or eSpCas9 1.1 — were selected for further analysis after the researchers scored the variants based on both efficiency and specificity.

Improved specificity could help CRISPR/Cas9 as it inches towards clinical use. "Many of the safety concerns are related to off-target effects [of CRISPR/Cas9]," Zhang said in a statement. "We hope the development of eSpCas9 will help address some of those concerns, but we certainly don't see this as a magic bullet. The field is advancing at a rapid pace, and there is still a lot to learn before we can consider applying this technology for clinical use."

In addition to improving certain applications of CRISPR/Cas9 genome editing, the authors said their study offered broad insight into the mechanics of CRISPR/Cas9 systems. "We propose that off-target cutting occurs when the strength of Cas9 binding to the non-target DNA strand exceeds forces of DNA re-hybridization," they wrote, adding that their model implied that specificity can decrease with stronger interactions between Cas9 and the non-target strand, though they did not say why one would want that to happen.

The scientists also said their strategy could be applied to improve the specificity of Cas9 enzymes from other bacterial species researchers might use in genome editing applications, such as Cas9 from Staphylococcus aureus and even other CRISPR enzymes such as Cpf1.