Saturation Mutagenesis Using Dual Cytosine and Adenine Base Editors

doi:10.11983/CBB21009

Abstract

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

Rui Zhang, Caixia Gao. Saturation Mutagenesis Using Dual Cytosine and Adenine Base Editors[J]. Chinese Bulletin of Botany, 2021, 56(1): 50-55.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB21009

https://www.chinbullbotany.com/EN/Y2021/V56/I1/50

Figures/Tables 2

Figure 1 Architectures of the binary vectors (A) pH-STEME-1-esgRNA; (B) pH-STEME-NG-esgRNA. OsU3p: Rice OsU3 promoter; Ubip: Maize Ubi-1 promoter; A3A: Cytidine deaminase APOBEC3A; ecTadA-ecTadA7.10: Adenosine deaminase ecTadA-ecTadA7.10; NLS: Nuclear localization signal; UGI: Uracil DNA glycosylase inhibitor; E9t: Pea rbcS-E9 terminator; 35Sp: Cauliflower mosaic virus 35S promoter; HygR: Hygromycin resistance gene; CaMVt: Cauliflower mosaic virus terminator; LB: T-DNA left border repeat; RB: T-DNA right border repeat; L: Linker sequence

Figure 2 Schematic of the procedure for targeted random mutagenesis and illustration of design for sgRNA pool (A) Schematic of the procedure for targeted random mutagenesis; (B) Illustration of design for sgRNA pool. Chose the target region of 3rd and 4th exons (E3: 270 bp and E4: 330 bp), then design 60 sgRNAs divided into 4 groups. sgRNAs in group 1 and 2 target NGD and HCN PAM sequences and the target region (700 bp) can be amplified using primers E3-F/E3-R. sgRNAs in group 3 and 4 target NGD and HCN PAM sequences and the target region (700 bp) can be amplified using primers E4-F/E4-R. Amplicons are about 700 bp and suitable for T7E1 assay.

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