Engineered CRISPR-Cas9 nucleases with altered PAM specificities

By B. Kleinstiver, Michelle S. Prew, S. Tsai, V. Topkar, Nhu T. Nguyen, Zongli Zheng, Andrew P. W. Gonzales, Zhuyun Li, Randall T. Peterson, J. Yeh and 6 more. 2015

1,453 citations recorded by the source corpus.

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

Subfields: Molecular Biology, Public Health, Environmental and Occupational Health

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Citations in the corpus

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (2018, neutral): “Primers and templates used in the synthesis of all sgRNA plasmids used in this work are listed in Supplementary Tables 8, 9, 11, 12, 15, and 16.”
Crystal structure of Cpf1 in complex with guide RNA and target DNA (2016, background): “Structure-guided engineering and direct evolution approaches have led to variants of Cas9 with enhanced target specificity (Slaymaker et al., 2016; Kleinstiver et al., 2016) or altered PAM requirements (Kleinstiver et al., 2015a, 2015b).”
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).”
Whole organism lineage tracing by combinatorial and cumulative genome editing (2016, background): “Finally, sgRNA sequences 357 and lengths (31), Cas9 cleavage activity and target preferences (32, 33), and the means by which 358 Cas9 and sgRNA(s) are expressed (e.”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “Several Cas9 proteins from differing species have been identified and used for gene editing, including Staphylococcus aureus (SaCas9) (Ran et al., 2013), Neisseria meningitidis (NmCas9) (Hou et al., 2013), and S. thermophiles (St1Cas9) (Kleinstiver et al., 2015).”
Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions (2017, methodology): “Joung and coworkers recently reported three SpCas9 mutants that accept NGA (VQR-Cas9), NGAG (EQR-Cas9), or NGCG (VRER-Cas9) PAM sequences 15 as well as an engineered SaCas9 variant containing three mutations (SaKKH-Cas9) that relax its PAM requirement to NNNRRT 16 .”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, background): “The molecular evolution of existing Cas9s has also led to the creation of new versions of Cas9 with altered PAM sequences (95, 96), thus expanding the targetable space within the mammalian genome.”
Identifying and visualizing functional PAM diversity across CRISPR-Cas systems (2016, methodology): “Previous depletion-based screens also identified a few functional PAMs that deviate from the consensus sequence (Kleinstiver et al., 2015).”