基于同源重组的 CRISPR 精准基因编辑技术 及其在农作物育种中的应用

随着 CRISPR 基因编辑技术的出现，几乎在任何动植物细胞基因组的特定目标位点，DNA 大片段 的“无缝”插入或替换，均可在 CRISPR 核酸酶产生双链切口后，在供体 DNA 存在的情况下，诱导同源定向修 复来实现。目前，这种基于同源重组的 CRISPR 精准基因编辑在农作物基因功能分析和新技术育种中正发挥着 越来越重要的作用。围绕在植物细胞中高效实现同源重组介导的 CRISPR 精准编辑这一目标，简述 CRISPR 精 准编辑依赖的两种主要的基于同源重组的细胞修复机制，即合成依赖的链退火修复机制和非同源末端连接辅助 的单链退火修复机制；在此基础上，详述产生 DNA 双链切口并诱导同源重组定向修复的 CRISPR 核酸酶和供体 DNA/RNA，主要包括 Cas9/12 及其融合蛋白、sgRNA/crRNA 及其修饰物、供体 DNA/RNA 及其修饰物；进而总 结在植物遗传转化中为保障 DNA 双链切口和供体 DNA/RNA 发生的时空一致性以提高同源重组效率，而通常采 用的 CRISPR 组分及供体 DNA/RNA 细胞递送方式；最后从功能基因组学研究和农作物新技术育种等方面，展望 基于同源重组的 CRISPR 精准基因编辑技术的应用前景。

Homologous Recombination-based CRISPR Precision Gene Editing Technology and Its Application in Crop Breeding

With the advent of CRISPR technologies, almost at any specific locus in any genome of animal or plant, large-size DNA fragment can be inserted or replaced seamlessly following the occurrence of CRISPR nuclease-produced double-strand break (DSB) and donor DNA, and the induction of homology-directed repair (HDR) can be realized. At present, the homologous recombination (HR)-based CRISPR precision gene editing plays increasingly important roles in crop gene functional analysis and new techniques breeding. In this review, aiming at effective realization of HR-based CRISPR precision gene editing in plant cells, we briefly review the two HR-based mechanisms underlying CRISPR precision gene editing, i.e., synthesis-dependent strand annealing (SDSA) and non-homologous end joining (NHEJ) assisted single strand annealing (SSA). Based on this, we elaborate the DSB-producing and HDR-inducing CRISPR nucleases and DNA/RNA donors, mainly including Cas9/12 and fusion proteins, sgRNA/crRNA and modifiers, and donor DNA/ RNA and modifiers. Furthermore, we summarize the adopted delivery systems of CRISPR components and donor DNA/ RNA during plant genetic transformation, in light of keeping spatiotemporal consistency of DSB and donor DNA/RNA to improve HR efficiency. Finally, we provide an outlook for the promising applications in crop functional genomics study and new techniques breeding of HRbased CRISPR precision gene editing.