Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has been demonstrated to be a robust genome engineering tool in a variety of organisms including plants. However, it has been shown that the CRISPR/Cas9 system cleaves genomic DNA seq...

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Main Authors:	Endo, Masaki, Mikami, Masafumi, Toki, Seiichi
Format:	Online
Language:	English
Published:	Oxford University Press 2015
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301742/

id	pubmed-4301742
recordtype	oai_dc
spelling	pubmed-43017422015-02-03 Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice Endo, Masaki Mikami, Masafumi Toki, Seiichi Rapid Paper The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has been demonstrated to be a robust genome engineering tool in a variety of organisms including plants. However, it has been shown that the CRISPR/Cas9 system cleaves genomic DNA sequences containing mismatches to the guide RNA strand. We expected that this low specificity could be exploited to induce multihomeologous and multiparalogous gene knockouts. In the case of polyploid plants, simultaneous modification of multiple homeologous genes, i.e. genes with similar but not identical DNA sequences, is often needed to obtain a desired phenotype. Even in diploid plants, disruption of multiparalogous genes, which have functional redundancy, is often needed. To validate the applicability of the CRISPR/Cas9 system to target mutagenesis of paralogous genes in rice, we designed a single-guide RNA (sgRNA) that recognized 20 bp sequences of cyclin-dependent kinase B2 (CDKB2) as an on-target locus. These 20 bp possess similarity to other rice CDK genes (CDKA1, CDKA2 and CDKB1) with different numbers of mismatches. We analyzed mutations in these four CDK genes in plants regenerated from Cas9/sgRNA-transformed calli and revealed that single, double and triple mutants of CDKA2, CDKB1 and CDKB2 can be created by a single sgRNA. Oxford University Press 2015-01 2014-11-11 /pmc/articles/PMC4301742/ /pubmed/25392068 http://dx.doi.org/10.1093/pcp/pcu154 Text en © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Endo, Masaki Mikami, Masafumi Toki, Seiichi
spellingShingle	Endo, Masaki Mikami, Masafumi Toki, Seiichi Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice
author_facet	Endo, Masaki Mikami, Masafumi Toki, Seiichi
author_sort	Endo, Masaki
title	Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice
title_short	Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice
title_full	Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice
title_fullStr	Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice
title_full_unstemmed	Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice
title_sort	multigene knockout utilizing off-target mutations of the crispr/cas9 system in rice
description	The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has been demonstrated to be a robust genome engineering tool in a variety of organisms including plants. However, it has been shown that the CRISPR/Cas9 system cleaves genomic DNA sequences containing mismatches to the guide RNA strand. We expected that this low specificity could be exploited to induce multihomeologous and multiparalogous gene knockouts. In the case of polyploid plants, simultaneous modification of multiple homeologous genes, i.e. genes with similar but not identical DNA sequences, is often needed to obtain a desired phenotype. Even in diploid plants, disruption of multiparalogous genes, which have functional redundancy, is often needed. To validate the applicability of the CRISPR/Cas9 system to target mutagenesis of paralogous genes in rice, we designed a single-guide RNA (sgRNA) that recognized 20 bp sequences of cyclin-dependent kinase B2 (CDKB2) as an on-target locus. These 20 bp possess similarity to other rice CDK genes (CDKA1, CDKA2 and CDKB1) with different numbers of mismatches. We analyzed mutations in these four CDK genes in plants regenerated from Cas9/sgRNA-transformed calli and revealed that single, double and triple mutants of CDKA2, CDKB1 and CDKB2 can be created by a single sgRNA.
publisher	Oxford University Press
publishDate	2015
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301742/
_version_	1613179060040499200

Multigene Knockout Utilizing Off-Target Mutations of the CRISPR/Cas9 System in Rice

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