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A Modular Cloning Toolkit for Genome Editing in Cereals

Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The CRISPR/Cas technology has recently become the tool of choice for gene editing applications in plants. Here we describe the method of assembling CRISPR/Cas DNA constructs, using the modular cloning (MoClo), for deployment in cereal species. For this purpose, we use a cloning toolkit that consists of a library of Golden Gate (GG) modules, which encode genetic elements, such as promoters, coding sequences of CRISPR/Cas nucleases (e.g. Cas9), terminators, guide RNA backbones, etc. A part of the toolkit are modules that allow assembly of tRNA-sgRNA polycistronic units without a PCR step involved. The tRNA-sgRNA system has proved to be efficient in a number of plant species, including cereals (rice, wheat). The cloning toolkit we present can be used for a variety of gene editing applications, including gene deletions and single nucleotide substitutions. It can also be easily expanded by adding new standardised parts.

Key words

  • CRISPR
  • Cas9
  • Plant
  • Genome editing
  • Golden Gate
  • MoClo
  • Cereal
  • Wheat

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Acknowledgement

Vladimir Nekrasov (Rothamsted Research) receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) Designing Future Wheat (DFW) programme.

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Correspondence to Vladimir Nekrasov .

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Hahn, F., Sanjurjo Loures, L., Nekrasov, V. (2022). A Modular Cloning Toolkit for Genome Editing in Cereals. In: Bilichak, A., Laurie, J.D. (eds) Accelerated Breeding of Cereal Crops. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1526-3_10

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  • DOI: https://doi.org/10.1007/978-1-0716-1526-3_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1525-6

  • Online ISBN: 978-1-0716-1526-3

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