Establishment of an efficient and
PAM
‐relaxed
LbCas12a
genome editing tool in plants
Yaolong Wang, Huanhuan Xu, Wenjun Lu, Zhiwen Wan, Ziyan Xu, Wenlong Wang, Chengyu Chen, Eryang Pan, Fangling Jiang, Tongkun Liu, Ying Li, Dong Xiao, Xuedong Yang, Fangfang Li, Xilin Hou, Changwei Zhang Summary
Cas12a is widely used in plant genome editing, but its targeting scope is constrained by stringent protospacer adjacent motif (PAM) requirements and variable activity across species, limiting its application at diverse genomic loci. LbCas12a‐RRV‐based editing system was established in nonheading Chinese cabbage, and T5exo‐PF‐LbCas12a was generated by introducing a triple mutation (D535G/S551F/D665N) and fusing with T5 exonuclease. This engineered system recognizes an expanded PAM sequence from 5′‐VTTV‐3′ to 5′‐NYHV‐3′. The system exhibited efficient editing at noncanonical PAM sites in cabbage, tomato, and rice. Additionally, it successfully mediated large‐fragment deletions via microhomology‐mediated end joining (MMEJ) in plants. This study expands Cas12a targeting scope in plants and provides the first evidence for Cas12‐mediated MMEJ‐based large‐fragment deletion. The toolkit facilitates functional genomics and crop improvement, and the methodology is readily adaptable to other plant species.