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Biallelic Gene Targeting in Rice.

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  • 1Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan (M.E., M.M., S.T.);Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, Kanagawa, 236-0027, Japan (M.M., S.T.); andKihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Yokohama, Kanagawa, 244-0813, Japan (S.T.)Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan (M.E., M.M., S.T.); Graduate School of Nanobioscience, Yokohama City University, 22-2, Seto, Kanazawa-ku, Yokohama, Kanagawa, 236-0027, Japan (M.M., S.T.); and Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Yokohama, Kanagawa, 244-0813, Japan (S.T.).

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Summary

Achieving gene targeting in plants via homology-directed repair (HDR) is challenging. This study found that targeting DNA ligase 4 significantly enhanced gene targeting efficiency in rice, overcoming previous limitations.

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Area of Science:

  • Plant biotechnology
  • Molecular biology
  • Genetics

Background:

  • Homology-directed repair (HDR)-mediated gene targeting (GT) using sequence-specific nucleases (SSNs) is established in many organisms.
  • Implementing HDR-mediated GT in plants is hindered by inefficient delivery of SSNs and HDR templates into plant nuclei.
  • Agrobacterium-mediated transformation is a preferred method for plant transformation, enabling delivery of large DNA payloads.

Purpose of the Study:

  • To improve the efficiency of Agrobacterium-mediated homology-directed repair (HDR) for gene targeting (GT) in rice.
  • To synchronize the induction of DNA double-strand breaks (DSBs) with the delivery of HDR templates in rice calli.
  • To investigate methods for enhancing gene targeting efficiency in plants.

Main Methods:

  • Rice calli were transformed with a Cas9 expression construct and a GT vector containing an HDR template and guide RNAs (gRNAs) targeting the acetolactate synthase (ALS) gene.
  • DSBs were induced in the ALS locus using the Cas9/gRNA complex, stimulating homologous recombination.
  • The effect of transiently expressed gRNAs and the prior transformation of gRNA targeting DNA ligase 4 on GT efficiency were evaluated.

Main Results:

  • Transient transcription of gRNAs from T-DNA containing the HDR template induced DSBs and stimulated homologous recombination in the ALS locus.
  • No significant difference in GT efficiency was observed between Cas9-expressing and non-expressing cells.
  • Co-transformation of gRNA targeting DNA ligase 4 with Cas9 dramatically increased GT efficiency, yielding multiple lines with biallelic GT at the ALS locus.

Conclusions:

  • Synchronizing DSB induction with HDR template delivery is crucial for efficient gene targeting in plants.
  • Targeting DNA ligase 4 represents a potent strategy to enhance gene targeting efficiency in rice.
  • This approach offers a promising avenue for improving plant genetic engineering techniques.