Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice

Abstract

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.

Keywords: CDK; CRISPR/Cas9; Off-target mutation; Rice.

Fig. 1

Schematic representation of CRISPR/Cas9-mediated target mutagenesis in this study. (A) Target site of CRISPR/Cas9-mediated target mutagenesis in the rice CDKB2 gene. The PAM sequence (NGG) is shown in blue and the 20 bp target sequence is shown in red. The red arrowhead indicates the expected cleavage site. (B) Homology of target sequences in CDKA and CDKB genes. Mismatches to the target sequence on CDKB2 are shown in pink. The dotted box indicates the BsiWI recognition sequence (CGTACG). (C) Vector constructs used in this study. pZH_OsCas9 was used for the first transformation and pZK_sgCDKB2 was used for the second transformation.

Fig. 2

Detection of mutations in the CDKB2 gene in Cas9-, sgRNA-expressing calli. (A) CAPS analysis of the CDKB2 locus. DNA extracted from independent pZK_sgCDKB2-transformed calli was subjected to PCR and subsequent BsiWI restriction enzyme digestion. M, marker; –RE, PCR product without restriction enzyme reaction; WT, BsiWI-digested PCR product of wild-type rice DNA. (B) Representative sequences of mutant alleles identified from Cas9-, sgRNA-expressing calli. The wild-type sequence is shown at the top with the PAM sequence highlighted in cyan and the target sequence in red. Dashes indicate deleted bases. The net change in length is noted to the right of each sequence (+, insertion; – deletion). The number of clones representing each mutant allele is shown in brackets. (C) Schematic representation of the chimeric state of multiply mutated and non-mutated cells in clonally propagated transgenic callus. The high proportion of mutated cells makes it easy to obtain mutated plants. Because mutations occur independently in single cells, various mutants can be obtained from a single Cas9, sgRNA transgenic line.

Fig. 3

On- and off-target mutation in regenerated plants. (A) CAPS analysis of the CDKB2, CDKA2 and CDKB1 loci. DNA extracted from independent regenerated plants obtained from pZH_OsCas9, pZK_sgCDKB2-transformed callus #17 was subjected to PCR and subsequent BsiWI restriction enzyme digestion. (B) Representative sequences of mutant alleles identified from regenerated plants. Mismatched bases to the target sequence are highlighted in blue in the wild-type sequence. Substituted and inserted bases are highlighted in orange.