Robust Genome Editing with Broad Targeting CRISPR Enzymes

Melanie Gonick

Programmable CRISPR enzymes are powerful and versatile tools for genome editing. They, however, require a specific protospacer adjacent motif (PAM) flanking the target site, which constrains the accessible sequence space for position-specific genome editing applications, such as base editing and precise gene insertion. For example, the standard Cas9 from Streptococcus pyogenes (SpyCas9) requires a PAM sequence of 5'-NGG-3' downstream of its RNA-programmed target, which limits genome editing applications to around 10% of all DNA sequences.

To broaden the targeting range of CRISPR, we first bioinformatically discover and characterize a highly similar SpyCas9 homolog from Streptococcus canis (ScCas9) with a more minimal 5’-NNG-3’ PAM specificity (Chatterjee, et al. Science Advances, 2018). Furthermore, we employ motifs from closely-related Streptococcus orthologs to engineer an optimized variant of ScCas9 (Sc++) that simultaneously exhibits broadened targeting capability, robust DNA cleavage activity, and minimal off-targeting propensity (Chatterjee, et al. Nature Biotechnology, 2020). Next, we recombine the PAM-interacting domain of Streptococcus macacae Cas9 (SmacCas9) with SpyCas9, and subsequently introduce enhancing mutations to generate iSpyMac with efficient and accurate 5’-NAA-3’ PAM preference (Chatterjee, et al. Nature Communications, 2020).  Together, these efforts expand the range of CRISPR nucleases to over 70% of DNA sequences, allowing for targeting of genomic loci that were previously inaccessible, including sequences within candidate genes for denser CRISPR screens and disease-related mutations that can now be fixed with genome editing architectures expressing our engineered variants.