Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform

Genome Biology

Research

Myriam Shafie¹  Frank Meulewaeter¹  Katelijn D’Halluin¹  David De Vleesschauwer¹  Jonas De Saeger²  Mattias Vermeersch²  Ward Develtere²  Christophe Gaillochet²  Thomas B. Jacobs²  Alexandra Peña Fernández²  Camila Gonzalez²  Andrzej Drozdzecki³  Vera Goossens³  Dominique Audenaert³  Julie Van Duyse⁴  Gert Van Isterdael⁴ 

[1] BASF Belgium Coordination Center CommV, Innovation Center Gent, Technologiepark-Zwijnaarde 101, 9052, Ghent, Belgium;Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark-Zwijnaarde 71, 9052, Ghent, Belgium;VIB Center for Plant Systems Biology, Technologiepark-Zwijnaarde 71, 9052, Ghent, Belgium;Screening Core, VIB, Technologiepark-Zwijnaarde 71, 9052, Ghent, Belgium;Centre for Bioassay Development and Screening (C-BIOS), Ghent University, Technologiepark-Zwijnaarde 71, 9052, Ghent, Belgium;VIB Flow Core, Technologiepark-Zwijnaarde 71, 9052, Ghent, Belgium;

关键词: CRISPR;    Plant biotechnology;    High-content imaging;    Cas9;    Cas12a;    Base editing;    Protoplasts;   

DOI  :  10.1186/s13059-022-02836-2

 received in 2022-05-06, accepted in 2022-12-06,  发布年份 2022

来源: Springer

【 摘 要 】

BackgroundTesting an ever-increasing number of CRISPR components is challenging when developing new genome engineering tools. Plant biotechnology has few high-throughput options to perform iterative design-build-test-learn cycles of gene-editing reagents. To bridge this gap, we develop ITER (Iterative Testing of Editing Reagents) based on 96-well arrayed protoplast transfections and high-content imaging.ResultsWe validate ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors (ABEs), allowing one optimization cycle — from design to results — within 3 weeks. Given that previous LbCas12a-ABEs have low or no activity in plants, we use ITER to develop an optimized LbCas12a-ABE. We show that sequential improvement of five components — NLS, crRNA, LbCas12a, adenine deaminase, and linker — leads to a remarkable increase in activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirm the activity of LbCas12a-ABE at endogenous targets in protoplasts and obtain base-edited plants in up to 55% of stable wheat transformants and the edits are transmitted to T1 progeny. We leverage these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE demonstrating that the optimizations can be broadly applied to the Cas12a toolbox.ConclusionOur data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants. We use ITER to create an efficient Cas12a-ABE by iteratively testing a large panel of vector components. ITER will likely be useful to create and optimize genome editing reagents in a wide range of plant species.

【 授权许可】

CC BY   
© The Author(s) 2022

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