Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

By J. H. Hu, Shannon M Miller, Maarten H. Geurts, Weixin Tang, Liwei Chen, N. Sun, Christina M. Zeina, Xue Gao, H. Rees, Zhi Lin and 1 more. 2018

1,381 citations recorded by the source corpus.

A key limitation of the use of the CRISPR–Cas9 system 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), 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 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 necessary trade-off between Cas9 editing efficiency, PAM compatibility and DNA specificity.

Subfields: Molecular Biology, Computational Biology & Bioinformatics

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Citations in the corpus

Biological structure and function emerge from scaling unsupervised learning to 250 million protein sequences (2019, background): “…the introduction of mutations to enhance protein function (Romero & Arnold, 2009), inform protein design using a combination of activating mutants (Hu et al., 2018), and make rational substitutions to optimize protein properties such as substrate speciﬁcity (Packer et al., 2017), stability (Tan…”
CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture. (2019, positive): “To expand the scope of targeting, Cas9 has been engineered to recognize different PAMs, such as VQR-Cas9 (NGA PAM), EQR-Cas9 (NGAG PAM), VRERCas9 (NGCG PAM), SaKKH-Cas9 (NNNRRT PAM) (61), xCas9 (NG, GAA, and GTA PAM) (44), and SpCas9-NG (NG PAM) (120).”
Evaluation and Minimization of Cas9-Independent Off-Target DNA Editing by Cytosine Base Editors (2020, positive): “We tested if these deaminases are compatible with SpCas9-NG, one of two recently reported Cas9 variants that recognize a broadened NG PAM 41,42 and found that YE1, and to a lesser extent YE2, YEE, EE, and R33A+K34A, maintained compatibility with SpCas9-NG nickase (Fig.”
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CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future (2020, background): “Several other variants of SpCas9 have also been engineered for broadening the gene target window including SpCas9-NG, which recognizes a minimal NG PAM (44) and xCas9, which recognizes a broad range of PAM including NG, GAA, and GAT (43).”