A Procedure to Design Guide RNA, Assemble Fragments, and Detect Mutation for Genome Editing in Flax

Part of the Springer Protocols Handbooks book series (SPH)


The game-changing molecular tool CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) has recently been developed as an effective genome-editing tool. It is used for targeted mutagenesis, whereby Cas9 enzyme creates a DNA double-strand break (DSB), which is then repaired by mutation-prone non-homologous end joining (NHEJ) repair system; consequently, the resulting DNA sequence collects mutations either through insertion or deletion of nucleotides. As there are already substantial challenges in the agricultural production, we are in need of highly efficient molecular tool such as CRISPR-Cas9 with detailed information so that the tool can be embraced and applied routinely. Flax (Linum usitatissimum L.) is one of the economically important crops; in this chapter, we provide a guideline to design guide RNA, assembly of DNA fragments, and a detailed protocol for protoplast isolation, transfection, and mutation detection.

Key words

CRISPR-Cas9 Genome editing CRISPRdirect Gibson assembly Flax 


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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of BiologyUniversity of British ColumbiaKelownaCanada
  2. 2.Universidade Federal Rural do Semi-aridoaCosta e SilvaBrazil
  3. 3.Department of Aquatic and Crop Resource DevelopmentNational Research Council of CanadaSaskatoonCanada

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