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 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.
Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan.
Masaki
M
Endo
Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan Graduate School of Nanobioscience, Yokohama City University, 22-2, Seto, Kanazawa-ku, Yokohama, Kanagawa, 236-0027 Japan.
Masafumi
M
Mikami
Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan Graduate School of Nanobioscience, Yokohama City University, 22-2, Seto, Kanazawa-ku, Yokohama, Kanagawa, 236-0027 Japan stoki@affrc.go.jp.
Seiichi
S
Toki
D001483:Base Sequence; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D018844:Cyclin-Dependent Kinases / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods*; D005818:Genetic Engineering; D016296:Mutagenesis; D009154:Mutation; D012275:Oryza / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics
D010940:Plant Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D018749:RNA, Plant; D018844:Cyclin-Dependent Kinases
CDK; CRISPR/Cas9; Off-target mutation; Rice