Characterization of diverse Cas9 orthologs for genome and epigenome editing

Since its discovery, CRISPR-Cas9 systems have shown tremendous promise as therapeutics, including the recent approval of the first Cas9-based therapeutic Casgevy for the treatment of sickle cell anemia and beta thalassemia. Butterfield, et al. of the Barrangou Lab at NC State used bioinformatics-based genome mining to identify novel Cas9 nucleases within the Lactobacillales genera. They sought to expand the toolbox of Cas9 proteins available for therapeutic use that can target new PAM sites and serve not just as nucleases, but as epigenetic regulators to activate or repress transcription of gene targets. The discovered Streptococcus Cas9 proteins were shown to be highly active as nucleases or as nuclease-null “dCas9” variants capable of genomic editing or epigenetic regulation in mammalian cells. myTXTL was used as a critical step in this research to discover the PAM site targeted by the novel Cas9 proteins through the use of an in vitro PAM discovery assay. This assay enabled the demonstration that the discovered Cas9 proteins targeted an A/T rich PAM site, greatly expanding the range of genomic targets of Cas9 which to date had relied on G/C rich PAM sites.