PDF(Pubmed)
Abstract：
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 Acidibacillus sulfuroxidans (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.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-024-00168-2.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s42994-024-00168-2.
摘要：
基因组编辑,特别是使用CRISPR/Cas系统，彻底改变了生物研究和作物改良。尽管CRISPR/Cas9的广泛使用，但它面临着诸如PAM序列要求和将其大蛋白递送到植物细胞中的挑战等限制。超小型Cas12f,衍生自酸性硫氧化杆菌（AsCas12f），由于其仅422个氨基酸的小尺寸以及对富含T的基序的偏好而脱颖而出，提供优于SpCas9的优势功能。然而,它的编辑效率在植物中非常低。最近的研究产生了两个AsCas12f变体，AsCas12f-YHAM和AsCas12f-HKRA,在哺乳动物细胞中展示更高的编辑效率，然而它们在植物中的表现仍未被探索。在这项研究中,通过对水稻基因组切割活性的系统研究，我们公布了两个AsCas12f变体的编辑效率的大幅提升，特别是对于AsCas12f-HKRA，实现了高达53%的编辑效率。此外，我们的分析表明，AsCas12f主要诱导靶DNA的缺失，显示出独特的缺失模式，主要集中在位置12、13、23和24，导致缺失大小主要为10和11bp，提示使用AsCas12f进行靶向DNA缺失的巨大潜力。这些发现扩展了植物高效基因组编辑的工具箱，为农业中的精确遗传修饰提供了有希望的前景。
■在线版本包含补充材料，可在10.1007/s42994-024-00168-2获得。
■在线版本包含补充材料，可在10.1007/s42994-024-00168-2获得。
