AUTHOR=An Yi , Geng Ya , Yao Junguang , Fu Chunxiang , Lu Mengzhu , Wang Chun , Du Juan 

TITLE=Efficient Genome Editing in Populus Using CRISPR/Cas12a

JOURNAL=Frontiers in Plant Science

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.593938

DOI=10.3389/fpls.2020.593938

ISSN=1664-462X

ABSTRACT=<p>The ability to create targeted mutations using clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 in support of forest tree biotechnology is currently limited. CRISPR/Cas12a is a novel CRISPR effector protein that not only broadens the CRISPR/Cas targeting range but also enables the generation of large-fragment deletions. In this study, a CRISPR/Cas12a system was evaluated for the induction of targeted mutations in the woody tree poplar (<italic>Populus alba</italic> × <italic>Populus glandulosa</italic>). Three Cas12a nucleases, namely, AsCas12a (<italic>Acidaminococcus</italic> sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (<italic>Francisella tularensis</italic> subsp. <italic>novicidain</italic> U112), were used. We knocked out multiple targets of the <italic>phytoene desaturase gene 8</italic> (<italic>PDS</italic>) using the CRISPR/Cas12a genome-targeting system, and the results indicated that the AsCas12a system is the most efficient. We further optimized the co-cultivation temperature after <italic>Agrobacterium</italic>-mediated transformation from 22 to 28°C to increase the Cas12a nuclease editing efficiency in poplar. AsCas12a showed the highest mutation efficiency, at 70%, and the majority of editing sites were composed of large-fragment deletions. By using this simple and high-efficiency CRISPR/Cas12a system, multiple targets can be modified to obtain multigene simultaneous knockout mutants in tree species, which will provide powerful tools with which to facilitate genetic studies of forest trees.</p>