Engineered CRISPR-Cas9 nucleases with altered PAM specificities

Abstract

Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. 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 analysis7. In addition, we identified and characterized 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 found 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.

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

“…Nuclease variants were generated by isothermal assembly into JDS246 (Addgene #43861) 5 , and guide RNAs were cloned into BsmB I-digested BPK1520 (Addgene #65777) 25 . Both U2OS cells (a gift from T. Cathomen, Freiburg) and U2OS-EGFP cells (encoding a single integrated copy of a pCMV-EGFP–PEST cassette) 26 were cultured at 37 °C with 5% CO 2 in advanced DMEM containing 10% heat-inactivated fetal bovine serum, 2 mM GlutaMax, penicillin/streptomycin, and 400 µg ml −1 Geneticin (for U2OS-EGFP cells only).…”

Whole organism lineage tracing by combinatorial and cumulative genome editing

“…Also, as we have shown, adjustments to the target sequences and dosages of editing reagents can be used to fine-tune mutation rates and to minimize undesirable intertarget deletions. Finally, sgRNA sequences and lengths (31), Cas9 cleavage activity and target preferences (32,33), and the means by which Cas9 and sgRNA(s) are expressed (e.g. transient, constitutive (34), or induced (35,36)), can be altered to control the pace, temporal window and tissue(s) at which the barcodes are mutated.…”

Genome-Editing Technologies: Principles and Applications

“…Similarly, fusing Cas9 to DNA-binding domains has also proven effective at improving its specificity (Bolukbasi et al 2015). Finally, several studies have recently showed that protein engineering can broadly enhance Cas9 specificity (Kleinstiver et al 2016;Slaymaker et al 2016) and even alter its PAM requirements (Kleinstiver et al 2015), the latter having the potential to enable creation of customized variants of Cas9 for allele-specific gene editing, although Cas9 orthologs Esvelt et al 2013;Hou et al 2013;Ran et al 2015) or alternative CRISPR systems (Zetsche et al 2015a) with unique PAM specificities have been uncovered in nature.…”