Establishment of an efficient and PAM-relaxed LbCas12a genome editing tool in plants.

IF 8.1 1区生物学 Q1 Agricultural and Biological Sciences

New Phytologist Pub Date : 2026-10-01 Epub Date: 2026-08-04 DOI:10.1111/nph.71498

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

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引用次数: 0

Abstract

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.

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一种高效且pam松弛的植物LbCas12a基因组编辑工具的建立

Cas12a广泛应用于植物基因组编辑，但其靶向范围受到严格的原间隔邻近基序（protospacer邻基序，PAM）要求和跨物种活性的限制，限制了其在不同基因组位点上的应用。以无头大白菜为材料，建立了基于lbcas12a - rrv的编辑系统，通过引入D535G/S551F/D665N三重突变，与T5外切酶融合产生t5exo - sf - lbcas12a。该工程系统可识别从5‘- vtv -3’到5‘-NYHV-3’的扩展PAM序列。该系统在白菜、番茄和水稻的非规范PAM位点显示出有效的编辑。此外，它还通过微同源介导的末端连接（microhomology-mediated end joining， MMEJ）在植物中成功介导了大片段缺失。本研究扩大了Cas12a在植物中的靶向范围，为cas12介导的基于mmej的大片段缺失提供了首个证据。该工具包促进了功能基因组学和作物改良，并且该方法很容易适用于其他植物物种。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

New Phytologist PLANT SCIENCES-

CiteScore

17.60

自引率

5.30%

发文量

728

审稿时长

1 months

期刊介绍： New Phytologist is a leading publication that showcases exceptional and groundbreaking research in plant science and its practical applications. With a focus on five distinct sections - Physiology & Development, Environment, Interaction, Evolution, and Transformative Plant Biotechnology - the journal covers a wide array of topics ranging from cellular processes to the impact of global environmental changes. We encourage the use of interdisciplinary approaches, and our content is structured to reflect this. Our journal acknowledges the diverse techniques employed in plant science, including molecular and cell biology, functional genomics, modeling, and system-based approaches, across various subfields.