Title: Robust Genome Editing with Broad-Targeting CRISPR Enzymes
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. 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. Next, we recombine the PAM-interacting domain of Streptococcus macacae Cas9 (SmacCas9) with SpyCas9, and subsequently introduce enhancing mutations to generate iSpyMac with altered and efficient 5’-NAA-3’ PAM preference. 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.
Joseph M. Jacobson, PhD, Associate Professor of Media Arts and Sciences, MIT (Advisor)
Benjamin P. Kleinstiver, PhD, Assistant Professor of Pathology, Massachusetts General Hospital
Kevin M. Esvelt, PhD, Assistant Professor of Media Arts and Sciences, MIT
George M. Church, PhD, Professor of Genetics, Harvard Medical School