PAM 유연성을 갖는 Un1Cas12f1 단백질 변이체 개발

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR- associated protein (Cas) system, notably Cas9, is hindered by its substantial size, which poses challenges for efficient cellular delivery and clinical applications. The large size of Cas9, particularly in the context of gene therapy using viral vectors like adeno-associated virus (AAV) with payload capacity constraints (<4.7 kb), restricts its clinical utility. Therefore, research focusing on smaller Cas proteins that can overcome these limitations has become increasingly important. Recent developments in hypercompact programmable RNA-guided DNA nucleases suggest exciting prospects for broadening their utility in clinical settings. Un1Cas12f1, one of well-known variant of Cas12f1, demonstrates an affinity for TTTR protospacer adjacent motif (PAM) and the engineered Un1Cas12f1 variant, Un1Cas12f1-D143R/T147R/E151A, and engineered gRNA of Un1Cas12f1, GE4.1, enables highly efficient genome editing in living cells. However, the restricted TTTR PAM recognition of Un1Cas12f1 imposes limitations on its diverse applications. So, we conducted engineering to create Un1Cas12f1 variants with non-TTTR PAM recognition. In this study, We engineered Un1Cas12f1 by referencing the engineered site of CWCas12f1, which differs only in the initial 28 amino acid residues, to explore variants with altered PAM specificities. Initially, we characterized the PAM profile of the Un1Cas12f1-D143R/T147R/E151A using PAM determination assays (PAMDA), confirming its enhanced efficiency compared to wild-type Un1Cas12f1. To further investigate altered PAM specificities, we introduced mutations at the R163 and S160 positions into the efficient Un1Cas12f1-D143R/T147R/E151A and characterized the resulting variants using PAMDA, in vitro cleavage assays, and assessment of indels at endogenous target sites. Our findings identified variants such as RRA-R163Q, which enhances specificity for the canonical TTTR PAM, and RRA-S160K, which broadens PAM recognition. These variants show trends akin to CWCas12f, suggesting potential for further engineering. Our PAMDA findings offer new insights into Un1Cas12f1 and its variants, paving the way for future gene therapy research and applications. Key words: CRISPR, Cas12f1, CasMINI, GE4.1 gRNA, PAM engineering