Directed evolution of Cas9 for mammalian genome editing
AbstractThe ability to manipulate the genome has been a longstanding goal since the discovery of the central dogma of molecular biology. Such a technology would not only allow us to correct deleterious mutations for human therapeutics but would also be greatly enabling for understanding the function of a given gene. The simplicity of the Cas9 system, an RNA-guided endonuclease, has led to widespread adoption in many animal and plant species. The ability to separate the DNA-binding activity of Cas9 from its nuclease activity has also given us the ability to tether a number of DNA effector domains to Cas9 to further expand its repertoire of DNA modifications. Nonetheless, a number of key limitations still exist for the system such as the requirement that a protospacer adjacent motif be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), the required PAM sequence is NGG. No natural or engineered Cas9 variants that have been shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG.
Here we use 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 our knowledge among Cas9 proteins that are active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and cytidine and adenine base editing. Notably, 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 trade-off between Cas9 editing efficiency, PAM compatibility and DNA specificity. Comparing the mutations in xCas9 with SpCas9 gives us additional clues to the underlying mechanism of DNA recognition and cutting employed by the Cas9 enzyme and general principles for designing new Cas9 variants. PACE has also been proven to be a versatile system for Cas9 evolution and could play a major role in the goal of building a suite of Cas9 variants that would allow us to programmatically modify any genomic sequence at will with high specificity.
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