[Intellectual Contribution]

Establishment of an efficient CRISPR/Cas9 mediated genome editing system in rice

Masaki Endo, Seiichi Toki
Plant Genome Engineering Research Unit
We compared the mutation frequency in various Cas9, sgRNA constructs under the same experimental condition and established an efficient targeted mutagenesis system via CRISPR/Cas9 system in rice. Using the sgRNA designed on the conserved region, multiple paralogous genes were mutagenized by single sgRNA by on- and off-target cleavage. In addition, we revealed that extension of the culture period of rice calli expressing Cas9 and sgRNA is an effective approach for increasing mutation efficiency.
Keywords: rice, CRISPR/Cas9, genome editing


The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 system (CRISPR/Cas9) has been demonstrated to be a robust genome engineering tool in a variety of organisms. There have also been several reports of successful CRISPR/Cas9-mediated targeted mutagenesis in rice. However, comparison of each result doesn’t make sense because target genes, tissues used for transformation, and evaluation methods of mutation frequency differ in each report. So we compared the mutation frequency in various Cas9, sgRNA expression constructs under the same experimental condition and selected the one that showed predominant expression. Furthermore, we attempted to disrupt multiple paralogous genes by using off-target mutation of CRISPR/Cas9, which is often regarded as a disadvantage in using the CRISPR/Cas9 system. In addition, we analyzed the effect of culture period of calli expressing Cas9 and sgRNA to determine the factors affecting mutation frequency.
[Results and Discussion]
  1. We introduced Cas9 and sgRNA expression cassettes separately and sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences (Fig. 1A). As a result, the best combination of Cas9, sgRNA was determined and all-in-one vector of these constructs was confirmed to be useful for target mutagenesis of rice with high-efficiency (Fig. 1B).
  2. CRISPR/Cas9 system has some propensity for causing off-target mutations and it is often regarded as a disadvantage. We attempted to use this off-target mutation for inducing mutations in paralogous genes in rice. When sgRNA was designed on consensus sequence of 4 CDK genes, mutation frequency of on-target gene (CDKB2) and the most strong candidate (CDKA2) with 1-nt mismatch in 20bp target sequence were almost the same. However, as the number of mismatch increased and the position of mismatch come close to PAM sequence (NGG), mutation frequency was decreased (Fig. 2, CDKB1 and CDKA1).
  3. Mutation frequency largely depends on the target sequence (data not shown). However, extension of the culture period of calli expressing Cas9 and sgRNA was effective for increasing the ratio of mutated cell (Fig. 3).
[Future prospects]
  1. Because vector construction of CRISPR/Cas9 system is much easier than ZFNs and TALENs and mutation frequency of CRISPR/Cas9 is relatively high, CRISPR/Cas9 system will be the major artificial nuclease used for genome engineering. In plant, direct delivery of RNA to plant nuclei is difficult. Thus, utilization of appropriate expression constructs for both gRNA and Cas9 is important. The CRISPR/Cas9 vector established in our study has been proven in many laboratories to induce targeted mutagenesis efficiently in rice and will contribute molecular breeding of rice.
  2. Off-target mutation is often referred to as a disadvantage in using the CRISPR/Cas9 system. However, we showed that such off-target mutations can be used for knockout of multiple genes with high homology by single sgRNA. In case of the gene families in polyploid or diploid plants, disruption of multiple genes is necessary to obtain the desired phenotype. Our study showed that off-target mutations could be utilized for plant molecular breeding.

Fig. 1. Selection of appropriate Cas9 and sgRNA expression construct in rice. (A) Six Cas9 constructs and two sgRNA constructs were transformed into rice calli separately and sequentially. The best combination of Cas9 and sgRNA construct was determined and an all-in-one vector was constructed. (B) Using an all-in-one vector, targeted mutagenesis of drooping leaf (DL) gene was conducted. As a result, bi-allelic mutants with the drooping leaf phenotype were obtained efficiently.


Fig. 2. Multigene knockout utilizing off-target mutations.
sgRNA was designed on the consensus sequence of 4 CDK genes. Mutation frequency in CDKA2 gene, which has 1-nt mismatch at 18-nt from PAM sequence (NGG) was almost the same as that in the on-target gene, CDKB2. When the number of mismatch increases and the position of mismatch comes close to PAM sequence, mutation frequency decreased (CDKA1, CDKB1).


Fig. 3. Increment of mutation frequency by extension of culture period of Cas9 and sgRNA transformed calli. Mutation in DNAs extracted from one and two months cultured calli with the Cas9 and sgRNA transgenic expression construct was detected by CAPS analysis. In one month cultured calli, a few mutated PCR products were detected whereas in 2 months cultured calli, mutated PCR product was significantly increased.



  1. Endo M, Mikami M, Toki S (2015) Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice Plant and Cell Physiology 56 (1):41-47
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