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Broad Researchers Develop More Widely Compatible Cas9 Variant

NEW YORK (GenomeWeb) – Broad Institute researcher David Liu and his colleagues have developed a Cas9 variant that is capable of recognizing a broader range of protospacer adjacent motif (PAM) sequences than the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), while also achieving greater DNA specificity and substantially lower genome-wide off-target activity.

"A key limitation to the use of CRISPR-Cas9 proteins for genome editing and other applications is the requirement that a PAM be present at the target site. For SpCas9, the required PAM sequence is NGG," the authors wrote in Nature this week.

"No natural or engineered Cas9 variants shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG," they added. "Here we used phage-assisted continuous evolution (PACE) to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA, and GAT."

The researchers used the PACE process to evolve 14 xCas9 variants containing consensus mutations. Through a series of experiments, they then characterized which of those mutations would be necessary to play a role in mediating DNA sequence recognition and switching Cas9 from an open to a closed conformation upon target recognition.

They then tested if those mutations were compatible with xCas9 function in mammalian cells, by characterizing xCas9 variants for their activity and PAM compatibility in human cells in four contexts: transcriptional activation, genomic DNA cutting, cytidine base editing, and adenine base editing.

For genomic DNA cleavage, the team found that xCas9 "modestly outperformed" SpCas9 at NGG PAM sites, but showed "substantially higher" cleavage than SpCas9 at non-NGG PAM sites.

They also targeted endogenous genomic sites in HEK293T cells and measured indel formation by high-throughput sequencing, and found that on the four NGG PAM sites tested, xCas9 showed comparable activity to SpCas9. However, all four NGT PAM sites showed a 4.5-fold increase in indel formation with xCas9 than with SpCas9. The four NGA PAM sites averaged a 1.6-fold increase with xCas9 compared to SpCas9.

"Taken together, these results indicate that xCas9 nuclease mediates target gene disruption at NGG PAM sites with comparable efficiencies as wild-type SpCas9, but cleaves NG, GAA, and GAT PAM sites with substantially higher efficiencies than SpCas9," the authors wrote.

The researchers also substituted xCas9 into the third-generation base editor (BE3) developed by Liu, and found it resulted in "substantially improved" base editing over SpCas9. In fact, they noted, using xCas9–BE3 could, in principle, increase the percentage of 4,422 pathogenic SNPs in the ClinVar database that could be targeted by C-G to T-A base editing from 26 percent to 73 percent, when compared to SpCas9–BE3 editing. Likewise, xCas9–adenine base editing increases the fraction of 14,969 pathogenic ClinVar SNPs that could be targeted by A-T to G-C base editing from 28 percent to 71 percent.

And importantly, the team did not find that xCas9 increased off-target activity. Indeed, experiments showed that despite its broadened PAM compatibility, xCas9 has substantially lower genome-wide off-target activity at NGG PAM sites than SpCas9, as well as minimal off-target activity at non-NGG PAM site, the researchers noted.

"These results, together with the success of multiple independent efforts to create high-fidelity Cas9 variants, suggest that the DNA promiscuity of wild-type Cas9, which likely evolved to impede viral evasion, can readily be overcome by protein engineering or evolution," the authors wrote. "That xCas9 exhibits much higher DNA specificity than SpCas9 even though it was not explicitly selected for this property suggests that the off-target activity of wild-type SpCas9 may lie at a narrow fitness peak suitable for defending the much smaller bacterial genome but not optimal for genome editing in mammalian cells. These observations are therefore consistent with a model in which native SpCas9 is poised to become more specific, rather than less specific, upon mutation."