Ye Zhengyan, Zhang Yuanyan, He Shiqi, Li Shaokang, Luo Longjiong, Zhou Yanbiao, Tan Junjie, Wan Jianmin
Sanya Institute of Nanjing Agricultural University, State Key Laboratory of Crop Genetics & Germplasm Enhancement and Utilization, Province and Ministry Co-sponsored Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing, 210095 China.
Zhongshan Biological Breeding Laboratory, No. 50 Zhongling Street, Nanjing, 210014 China.
aBIOTECH. 2024 May 28;5(2):184-188. doi: 10.1007/s42994-024-00168-2. eCollection 2024 Jun.
Genome editing, particularly using the CRISPR/Cas system, has revolutionized biological research and crop improvement. Despite the widespread use of CRISPR/Cas9, it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells. The hypercompact Cas12f, derived from (AsCas12f), stands out due to its small size of only 422 amino acids and its preference for a T-rich motif, presenting advantageous features over SpCas9. However, its editing efficiency is extremely low in plants. Recent studies have generated two AsCas12f variants, AsCas12f-YHAM and AsCas12f-HKRA, demonstrating higher editing efficiencies in mammalian cells, yet their performance in plants remains unexplored. In this study, through a systematic investigation of genome cleavage activity in rice, we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants, particularly for AsCas12f-HKRA, which achieved an editing efficiency of up to 53%. Furthermore, our analysis revealed that AsCas12f predominantly induces deletion in the target DNA, displaying a unique deletion pattern primarily concentrated at positions 12, 13, 23, and 24, resulting in deletion size mainly of 10 and 11 bp, suggesting significant potential for targeted DNA deletion using AsCas12f. These findings expand the toolbox for efficient genome editing in plants, offering promising prospects for precise genetic modifications in agriculture.
The online version contains supplementary material available at 10.1007/s42994-024-00168-2.
基因组编辑，特别是使用CRISPR/Cas系统，已经彻底改变了生物学研究和作物改良。尽管CRISPR/Cas9被广泛使用，但它面临着诸如PAM序列要求等限制，以及将其大蛋白导入植物细胞的挑战。源自嗜热栖热菌（AsCas12f）的超紧凑型Cas12f因其仅422个氨基酸的小尺寸以及对富含T基序的偏好而脱颖而出，比SpCas9具有优势。然而，它在植物中的编辑效率极低。最近的研究产生了两个AsCas12f变体，AsCas12f-YHAM和AsCas12f-HKRA，它们在哺乳动物细胞中显示出更高的编辑效率，但其在植物中的性能仍未得到探索。在本研究中，通过对水稻基因组切割活性的系统研究，我们发现这两个AsCas12f变体的编辑效率都有显著提高，特别是AsCas12f-HKRA，其编辑效率高达53%。此外，我们的分析表明，AsCas12f主要诱导目标DNA的缺失，呈现出独特的缺失模式，主要集中在第12、13、23和24位，导致缺失大小主要为10和11 bp，这表明使用AsCas12f进行靶向DNA缺失具有巨大潜力。这些发现扩展了植物高效基因组编辑的工具库，为农业中的精确基因改造提供了广阔前景。
在线版本包含可在10.1007/s42994-024-00168-2获取的补充材料。
