A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants

Author:

Xiong Xiangyu1,Li Zhenxiang1ORCID,Liang Jieping1,Liu Kehui1,Li Chenlong1ORCID,Li Jian-Feng1ORCID

Affiliation:

1. MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China

Abstract

Abstract CRISPR/Cas-derived base editing tools empower efficient alteration of genomic cytosines or adenines associated with essential genetic traits in plants and animals. Diversified target sequences and customized editing products call for base editors with distinct features regarding the editing window and target scope. Here we developed a toolkit of plant base editors containing AID10, an engineered human AID cytosine deaminase. When fused to the N-terminus or C-terminus of the conventional Cas9 nickase (nSpCas9), AID10 exhibited a broad or narrow activity window at the protospacer adjacent motif (PAM)-distal and -proximal protospacer, respectively, while AID10 fused to both termini conferred an additive activity window. We further replaced nSpCas9 with orthogonal or PAM-relaxed Cas9 variants to widen target scopes. Moreover, we devised dual base editors with AID10 located adjacently or distally to the adenine deaminase ABE8e, leading to juxtaposed or spaced cytosine and adenine co-editing at the same target sequence in plant cells. Furthermore, we expanded the application of this toolkit in plants for tunable knockdown of protein-coding genes via creating upstream open reading frame and for loss-of-function analysis of non-coding genes, such as microRNA sponges. Collectively, this toolkit increases the functional diversity and versatility of base editors in basic and applied plant research.

Funder

National Transgenic Science and Technology Major Program of China

Foundation of Guangzhou Science and Technology Key Project

National Natural Science Foundation of China

China Postdoctoral Science Foundation

Publisher

Oxford University Press (OUP)

Subject

Genetics

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