基于双碱基编辑系统的植物基因靶向随机突变技术

Saturation Mutagenesis Using Dual Cytosine and Adenine Base Editors

遗传性变异是表型多样性的基础,靶向饱和突变作物基因可以促进产生具有优异农艺性状的突变体。相较于传统诱变育种和异源物种中的定向进化方法,基于双碱基编辑系统的植物基因靶向随机突变技术可对植物内源基因产生高效突变,从而实现原位定向进化,加快植物育种及功能基因研究进程。该文介绍了使用饱和靶向内源基因突变编辑器(STEME)对植物功能基因进行靶向随机突变的具体操作方法,从靶点设计和突变体检测方面进行了详细描述。

Because the genome of an organism determines its primary phenotype,evolutionary principles suggest that genetic variations enhance phenotypic diversity towards increased fitness.Targeted saturation mutagenesis of crop genes could be used to screen for genetic variants with improved agronomic traits.Compared to traditional mutational breeding or directed evolution in heterologous organisms,targeted mutagenesis via dual cytosine and adenine base editors effectively generates endogenous mutagenesis and facilitates in vivo directed evolution of plant genes.In this protocol,we detail the process towards using saturated targeted endogenous mutagenesis editors(STEMEs)to generate targeted,random mutagenesis of plant genes.In particular,we focus on the process of designing targets,screening and genotyping the resulting evolved variants.