Liu Shishi, He Yao, Fan Tingting, Zhu Meirui, Qi Caiyan, Ma Yanqin, Yang Mengqiao, Yang Liang, Tang Xu, Zhou Jianping, Zhong Zhaohui, An Xueli, Qi Yiping, Zhang Yong
Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Chongqing Key Laboratory of Tree Germplasm Innovation and Utilization, School of Life Sciences, Southwest University, Chongqing, China.
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, China.
Plant Biotechnol J. 2025 Jan;23(1):156-173. doi: 10.1111/pbi.14486. Epub 2024 Oct 10.
Class 2 Type V-A CRISPR-Cas (Cas12a) nucleases are powerful genome editing tools, particularly effective in A/T-rich genomic regions, complementing the widely used CRISPR-Cas9 in plants. To enhance the utility of Cas12a, we investigate three Cas12a orthologs-Mb3Cas12a, PrCas12a, and HkCas12a-in plants. Protospacer adjacent motif (PAM) requirements, editing efficiencies, and editing profiles are compared in rice. Among these orthologs, Mb3Cas12a exhibits high editing efficiency at target sites with a simpler, relaxed TTV PAM which is less restrictive than the canonical TTTV PAM of LbCas12a and AsCas12a. To optimize Mb3Cas12a, we develop an efficient single transcription unit (STU) system by refining the linker between Mb3Cas12a and CRISPR RNA (crRNA), nuclear localization signal (NLS), and direct repeat (DR). This optimized system enables precise genome editing in rice, particularly for fine-tuning target gene expression by editing promoter regions. Further, we introduced Arginine (R) substitutions at Aspartic acid (D) 172, Asparagine (N) 573, and Lysine (K) 579 of Mb3Cas12a, creating two temperature-tolerant variants: Mb3Cas12a-R (D172R) and Mb3Cas12a-RRR (D172R/N573R/K579R). These variants demonstrate significantly improved editing efficiency at lower temperatures (22 °C and 28 °C) in rice cells, with Mb3Cas12a-RRR showing the best performance. We extend this approach by developing efficient Mb3Cas12a-RRR STU systems in maize and tomato, achieving biallelic mutants targeting single or multiple genes in T lines cultivated at 28 °C and 25 °C, respectively. This study significantly expands Cas12a's targeting capabilities in plant genome editing, providing valuable tools for future research and practical applications.
2类V-A型CRISPR-Cas（Cas12a）核酸酶是强大的基因组编辑工具，在富含A/T的基因组区域中特别有效，可作为植物中广泛使用的CRISPR-Cas9的补充。为了提高Cas12a的实用性，我们在植物中研究了三种Cas12a直系同源物——Mb3Cas12a、PrCas12a和HkCas12a。在水稻中比较了原间隔序列临近基序（PAM）要求、编辑效率和编辑谱。在这些直系同源物中，Mb3Cas12a在具有更简单、宽松的TTV PAM的靶位点上表现出高编辑效率，该PAM比LbCas12a和AsCas12a的经典TTTV PAM限制更少。为了优化Mb3Cas12a，我们通过优化Mb3Cas12a与CRISPR RNA（crRNA）、核定位信号（NLS）和同向重复序列（DR）之间的接头，开发了一种高效的单转录单元（STU）系统。这种优化的系统能够在水稻中进行精确的基因组编辑，特别是通过编辑启动子区域来微调靶基因的表达。此外，我们在Mb3Cas12a的天冬氨酸（D）172、天冬酰胺（N）573和赖氨酸（K）579处引入精氨酸（R）替代，创建了两个耐温变体：Mb3Cas12a-R（D172R）和Mb3Cas12a-RRR（D172R/N573R/K579R）。这些变体在水稻细胞中较低温度（22°C和28°C）下表现出显著提高的编辑效率，其中Mb3Cas12a-RRR表现最佳。我们通过在玉米和番茄中开发高效的Mb3Cas12a-RRR STU系统扩展了这种方法，分别在28°C和25°C培养的T系中实现了靶向单个或多个基因的双等位基因突变体。这项研究显著扩展了Cas12a在植物基因组编辑中的靶向能力，为未来的研究和实际应用提供了有价值的工具。
