(305e) Engineering CRISPR-Cas9 with Relaxed PAM Specificity for Gene Repression
AIChE Annual Meeting
2021
2021 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Design, Engineering, and Structural Prediction of Peptides and Proteins
Tuesday, November 9, 2021 - 1:42pm to 2:00pm
CRISPR-Cas9 nuclease has been robustly repurposed to build up the arsenal of tools for genome engineering and gene regulation in multiple types of cells. The most developed Cas9, Streptococcus pyogenes Cas9 (SpCas9), however, strictly recognizes 5â-NGG-3â PAM, which significantly limits its targetable DNA sequences. To apply SpCas9 as a universal repressor, we generated variants of SpCas9 with deactivated nuclease activity (SpdCas9) to adopt 5â-CAT-3â PAM so that the engineered SpdCas9 can bind to the most common start codon (AUG). By rationally engineering the PI (PAM interaction) domain of the previously developed SpdNG (5â-NG-3â PAM), we obtained a variant with expanded PAM range including the canonical 5â-NGG-3â and new 5â-CAT-3â PAM specificities. In vivo and in vitro cleavage assay confirmed the relaxed PAM range. The new SpdNG variant was further demonstrated as a tunable chromosomal gene repressor with more flexible sgRNA design. Our work showcased the feasibility of engineering a PAM-flexible Cas9 targeting signature non-G PAMs and created a novel SpdCas9-based transcriptional repressor.