Chinese Bulletin of Botany ›› 2021, Vol. 56 ›› Issue (1): 50-55.DOI: 10.11983/CBB21009

• INVITED PROTOCOLS • Previous Articles     Next Articles

Saturation Mutagenesis Using Dual Cytosine and Adenine Base Editors

Rui Zhang, Caixia Gao*()   

  1. Center for Genome Editing, State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing 100101, China
  • Received:2021-01-11 Accepted:2021-02-25 Online:2021-01-01 Published:2021-02-25
  • Contact: Caixia Gao

Abstract:

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.

Key words: base editing, targeted random mutagenesis, directed evolution of plant gene