Cargando…

Efficient Genome Editing in Populus Using CRISPR/Cas12a

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...

Descripción completa

Detalles Bibliográficos
Autores principales: An, Yi, Geng, Ya, Yao, Junguang, Fu, Chunxiang, Lu, Mengzhu, Wang, Chun, Du, Juan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720674/
https://www.ncbi.nlm.nih.gov/pubmed/33329659
http://dx.doi.org/10.3389/fpls.2020.593938
_version_ 1783619895367303168
author An, Yi
Geng, Ya
Yao, Junguang
Fu, Chunxiang
Lu, Mengzhu
Wang, Chun
Du, Juan
author_facet An, Yi
Geng, Ya
Yao, Junguang
Fu, Chunxiang
Lu, Mengzhu
Wang, Chun
Du, Juan
author_sort An, Yi
collection PubMed
description 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 (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) 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 Agrobacterium-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.
format Online
Article
Text
id pubmed-7720674
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-77206742020-12-15 Efficient Genome Editing in Populus Using CRISPR/Cas12a An, Yi Geng, Ya Yao, Junguang Fu, Chunxiang Lu, Mengzhu Wang, Chun Du, Juan Front Plant Sci Plant Science 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 (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) 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 Agrobacterium-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. Frontiers Media S.A. 2020-11-19 /pmc/articles/PMC7720674/ /pubmed/33329659 http://dx.doi.org/10.3389/fpls.2020.593938 Text en Copyright © 2020 An, Geng, Yao, Fu, Lu, Wang and Du. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
An, Yi
Geng, Ya
Yao, Junguang
Fu, Chunxiang
Lu, Mengzhu
Wang, Chun
Du, Juan
Efficient Genome Editing in Populus Using CRISPR/Cas12a
title Efficient Genome Editing in Populus Using CRISPR/Cas12a
title_full Efficient Genome Editing in Populus Using CRISPR/Cas12a
title_fullStr Efficient Genome Editing in Populus Using CRISPR/Cas12a
title_full_unstemmed Efficient Genome Editing in Populus Using CRISPR/Cas12a
title_short Efficient Genome Editing in Populus Using CRISPR/Cas12a
title_sort efficient genome editing in populus using crispr/cas12a
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720674/
https://www.ncbi.nlm.nih.gov/pubmed/33329659
http://dx.doi.org/10.3389/fpls.2020.593938
work_keys_str_mv AT anyi efficientgenomeeditinginpopulususingcrisprcas12a
AT gengya efficientgenomeeditinginpopulususingcrisprcas12a
AT yaojunguang efficientgenomeeditinginpopulususingcrisprcas12a
AT fuchunxiang efficientgenomeeditinginpopulususingcrisprcas12a
AT lumengzhu efficientgenomeeditinginpopulususingcrisprcas12a
AT wangchun efficientgenomeeditinginpopulususingcrisprcas12a
AT dujuan efficientgenomeeditinginpopulususingcrisprcas12a