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Hybrid Cas12a Variants with Relaxed PAM Requirements Expand Genome Editing Compatibility

ACS Synth Biol. 2024-05; 
Zhenyu Liu, Huayi Liu, Chaoqun Huang, Qun Zhou, Yunzi Luo
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Proteins, Expression, Isolation and Analysis … The supernatant was mixed with Ni-NTA agarose resin (GenScript, China) and loaded onto an affinity chromatography column (Sangon Biotech, China). The Ni-NTA resin was washed … Get A Quote

摘要

Cas12a is a widely used programmable nuclease for genome editing across a variety of organisms, but its application is limited by its PAM recognition restriction. To alleviate these PAM constraints, protein engineering efforts have been applied to expand the PAM recognition range. In this study, we designed and constructed 990 synthetic hybrid Cas12a chimeras through domain shuffling and screened an efficient hybrid Cas12a (ehCas12a) that could recognize a broad range PAM of 5'-TYYN-3' (Y is T or C and N is A, T, C, or G). Furthermore, we constructed an ehCas12a variant, ehCas12a RRVR (T167R/N572R/K578V/N582R), with expanded PAM preference to 5'-TNYN, TWRV-3' (W is A or T, R is A or G, and V is A, C, or G), whi... More

關鍵詞

Cas12a, PAM recognition, base editing, domain shuffling, synthetic biology
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