Structure-Guided Engineering of AceCas9 to Relax the PAM Requirement

The CRISPR-Cas9 system is a powerful defense mechanism that confers resistance to bacteriophage infection. Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C effector. AceCas9 currently cleaves DNA with a 5’-NNNCC-3’ protospacer adjacent motif (PAM) sequence and is sensitive to CpC cytosine methylation. Engineering different variants of AceCas9 will expand the range of applications for the protein. Specifically, mutating the protein to recognize a PAM sequence of 5’-NNNCG-3’ while maintaining methylation sensitivity allow us to utilize AceCas9 to detect samples with CpG methylations. Three residues, E1044, R1088, and R1091 play a very important role in PAM recognition in AceCas9. We created site-specific mutations on residues that confer PAM specificity to weaken the PAM requirement. The PAM interacting domain mutants on AceCas9 were insufficient at recognizing the PAM. AceCas9 contains a phosphate lock loop that interacts closely with the target strand phosphate near the PAM sequence. We utilized a bacterial cellular survival assay to discover that the phosphate lock loop plays a role in PAM specificity. Point mutations of E839R and E840Y (RY) allow AceCas9 to cleave a 5’-NNNAC-3’ PAM sequence. In addition, we added a point mutation of V709A to increase the catalytic activity of the RY mutant of AceCas9.