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Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice

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Abstract

Key message

Frequency of CRISPR/Cas9-mediated targeted mutagenesis varies depending on Cas9 expression level and culture period of rice callus.

Abstract

Recent reports have demonstrated that the CRISPR/Cas9 system can function as a sequence-specific nuclease in various plant species. Induction of mutation in proliferating tissue during embryogenesis or in germline cells is a practical means of generating heritable mutations. In the case of plant species in which cultured cells are used for transformation, non-chimeric plants can be obtained when regeneration occurs from mutated cells. Since plantlets are regenerated from both mutated and non-mutated cells in a random manner, any increment in the proportion of mutated cells in Cas9- and guide RNA (gRNA)-expressing cells will help increase the number of plants containing heritable mutations. In this study, we examined factors affecting mutation frequency in rice calli. Following sequential transformation of rice calli with Cas9- and gRNA- expression constructs, the mutation frequency in independent Cas9 transgenic lines was analyzed. A positive correlation between Cas9 expression level and mutation frequency was found. This positive relationship was observed regardless of whether the transgene or an endogenous gene was used as the target for CRISPR/Cas9-mediated mutagenesis. Furthermore, we found that extending the culture period increased the proportion of mutated cells as well as the variety of mutations obtained. Because mutated and non-mutated cells might proliferate equally, these results suggest that a prolonged tissue culture period increases the chance of inducing de novo mutations in non-mutated cells. This fundamental knowledge will help improve systems for obtaining non-chimeric regenerated plants in many plant species.

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Abbreviations

CAPS:

Cleaved amplified polymorphic sequences

Cas9:

CRISPR-associated endonuclease 9

CDKB2:

Cyclin-Dependent Kinase B2

CIM:

Callus induction medium

CRISPR:

Clustered regularly interspaced short palindromic repeat

gRNA:

Guide RNA

HMF:

Highest mutation frequency

NLS:

Nuclear localization signal

nt:

Nucleotide

PAM:

Protospacer adjacent motif

RT-PCR:

Reverse transcriptase-polymerase chain reaction

RC:

Ratio of calli with mutations

YSA:

Young seeding albino

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Acknowledgments

We thank K. Amagai, R. Aoto, A. Nagashii and F. Suzuki for general experimental technical support. This research was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation PGE1001) and the NIAS Strategic Research Fund. This work was also supported by the Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for creating next-generation agriculture, forestry and fisheries” (funding agency: Bio-oriented Technology Research Advancement Institution, NARO).

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Authors and Affiliations

  1. Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Yokohama, Kanagawa, 236-0027, Japan

    Masafumi Mikami & Seiichi Toki

  2. Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan

    Masafumi Mikami, Seiichi Toki & Masaki Endo

  3. Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Yokohama, Kanagawa, 244-0813, Japan

    Seiichi Toki

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  1. Masafumi Mikami
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Correspondence to Masaki Endo.

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Communicated by K. Toriyama.

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Mikami, M., Toki, S. & Endo, M. Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice. Plant Cell Rep 34, 1807–1815 (2015). https://doi.org/10.1007/s00299-015-1826-5

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  • DOI: https://doi.org/10.1007/s00299-015-1826-5

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