Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

Abstract

Summary A key limitation to the use of CRISPR-Cas9 proteins for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), this PAM requirement is NGG. No natural or engineered Cas9 variants shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. 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 PAM compatibility of xCas9 is the broadest reported to date among Cas9s active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and both cytidine and adenine base editing. Remarkably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility, and DNA specificity.

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…Thus, there is an urgent need for new engineered Cas nuclease which has a broadened editing range. Recently, a SpCas9 variant, xCas9, has been developed, which recognizes a PAM site as 5′‐NG‐3′ with increased fidelity . We envision that xCas9 may take the place of the canonical SpCas9.…”

CRISPR‐Cas system: Toward a more efficient technology for genome editing and beyond

“…Although point mutations are one of the most common sources of disease‐associated DNA, current technologies to site‐specific gene editing are inefficient and typically induce unwanted changes in the genome. In this perspective, scientists reported a new type of base editors (BEs) that offers potential improvements in efficiency (Table ) . The BE1 created by APOBEC1‐dCas9 fusion enhanced editing fidelity relevant to human disease.…”

Creating cell and animal models of human disease by genome editing using CRISPR/Cas9

“…Because it is not unexpected to find no PAM sequences around a target site (such as is the case for AT‐rich sites), it is therefore not possible to achieve precise genome editing using SpCas9. To overcome this limitation and increase the precision and specificity of Cas9, researchers have recently used phage‐assisted continuous evolution to design a Cas9‐based enzyme called xCas9, which can identify broad PAM sites such as GAA, NG and GAT …”