Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

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外文摘要：In plants and mammals,non-homologous end-joining is the dominant pathway to repair DNA double-strand breaks,making it challenging to generate knock-in events.In this study,we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana.We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems.In Arabidopsis thaliana,fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants.In addition,we demonstrated stable and heritable knock-ins in wheat in 1％of the primary transformants.Taken together,our results open perspectives for the routine production of her-itable knock-in and gene replacement events in plants.

外文关键词：

homology-directed repairknockingene replacementCRISPR-Cas5'exonucleaseplants

作者：

Tom Schreiber、Anja Prange、Petra Sch?fer、Thomas Iwen、Ramona Grützner、Sylvestre Marillonnet、Aurélie Lepage、Marie Javelle、Wyatt Paul、Alain Tissier

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作者单位：

Department of Cell and Metabolic Biology,Leibniz Institute of Plant Biochemistry,Weinberg 3,06120 Halle(Saale),Germany

Limagrain,Centre de Recherche,Route d'Ennezat,CS 90126,63720 Chappes,France

基金：

frame of the program"Crop plants of the future"from the Bundesministerium für Bildung und ForschungInvestissement d'Avenir program of the French National Agency of Research for the project GENIUSsubject of a Limagrain patent application

项目编号：

031B0548ANR-11-BTBR-0001_GENIUSWO2023/067020

出版年：

2024

DOI：

10.1016/j.molp.2024.03.013

分子植物(英文版)

中科院上海生命科学研究院植物生理生态所　中国植物生理学会

分子植物(英文版)

CSTPCD

影响因子：0.659

ISSN：1674-2052

年,卷(期)：2024.17(5)