Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
Graduate School of Nanobioscience, Yokohama City University, Kanagawa, Japan.
Methods Mol Biol. 2025;2869:75-90. doi: 10.1007/978-1-0716-4204-7_9.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR-Cas) is an adaptive prokaryote immune system against foreign DNA/RNA that is now applied widely to genome editing. A miniature Cas, CRISPR-Cas12f, is one-half to one-third the size of the CRISPR-Cas9 that is commonly used in genome editing experiments in many organisms, including higher plants. The compactness of CRISPR-Cas12f is expected to be advantageous in terms of vector construction and transformation frequency. Moreover, CRISPR-Cas12f can be useful for virus vector-mediated genome editing because the size of the transgene is the major restriction in the use of virus vectors. Here, we describe our protocol for targeted mutagenesis using Cas12f derived from Syntrophomonas palmitatica (SpCas12f) via Agrobacterium-mediated transformation in rice. We also summarize some approaches to improve the frequency of targeted mutagenesis using SpCas12f.
成簇规律间隔短回文重复序列 (CRISPR) 和 CRISPR 相关蛋白 (Cas) (CRISPR-Cas) 是一种针对外来 DNA/RNA 的适应性原核生物免疫系统，现在已广泛应用于基因组编辑。一种微型 Cas，CRISPR-Cas12f，是在许多生物体（包括高等植物）中常用于基因组编辑实验的 CRISPR-Cas9 的一半到三分之一大小。CRISPR-Cas12f 的紧凑性有望在载体构建和转化频率方面具有优势。此外，CRISPR-Cas12f 可用于病毒载体介导的基因组编辑，因为转基因的大小是使用病毒载体的主要限制因素。在这里，我们描述了通过农杆菌介导的转化在水稻中使用源自棕榈烯糖单胞菌 (Syntrophomonas palmitatica) 的 Cas12f 进行靶向诱变的方案。我们还总结了一些提高使用 SpCas12f 进行靶向诱变频率的方法。
