Abstract
Domain expansion contributes to diversification of RNA-guided-endonucleases including Cas9. However, it remains unclear how REC domain expansion could benefit Cas9. In this study, we identify an insertion spot that is compatible with large REC insertion and succeeds in enlarging the non-catalytic REC domain of Streptococcus pyogenes Cas9. The natural-evolution-like giant SpCas9 (GS-Cas9) is created and shows substantially improved editing precision. We further discover that enlarging the REC domain could enable regulation of the N-terminal adenine deaminase TadA8e tethered to the Cas9 scaffold, which contributes to substantially reducing unexpected editing and improving the precision of the adenine base editor ABE8e. We provide proof of concept for evolution-inspired expansion of Cas9 and offer an alternative solution for optimizing gene editors. Our study also indicates a vast potential for engineering the topological malleability of RNA-guided endonucleases and base editors.