PAMless SpRY recognizes a non-PAM region for efficient targeting

Abstract

Abstract Protospacer-adjacent motif (PAM) recognition licenses Cas nucleases for genome engineering applications, thereby restricting gene targets to regions containing PAMs. To overcome the PAM constraint, studies have applied protein engineering and successfully acquired PAM-relaxed SpCas9 variants, such as SpG (NGN PAMs) and SpRY (NRN>NYN PAMs). Given the evolved role of PAMs in facilitating target searching kinetics, it remains unclear how SpG and SpRY are able to quickly find their targets for cleavage after PAM relaxation. Here we combined experiments, simulations and theoretical analyses to investigate how SpG and SpRY search for their targets. Our results show that SpG and SpRY are spacer-dependent PAM-relaxed variants. To compensate for the relaxed PAM recognition, SpG and SpRY have been engineered to recognize position 1 or 1 to 3 of the target sequence through R61 and R1322, respectively. Moreover, our results demonstrate a significant decrease in target search kinetics for SpCas9 PAMless variants when PAM recognition is absent, with a slowdown of 3-4 orders of magnitude compared to wild-type SpCas9. This highlights the importance of considering Cas9 target searching kinetics in the successful engineering of PAMless SpCas9 variants. Overall, our results provide valuable insights for further PAMless Cas9 protein engineering efforts.