Abstract
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.
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Change history
22 October 2020
Correction to: Chapter 11 in: M. Tofazzal Islam, Pankaj K. Bhowmik and Kutubuddin A. Molla (eds.), CRISPR-Cas Methods, Springer Protocols Handbooks.
References
Mohanta T, Bashir T, Hashem A, Abd_Allah E, Bae H (2017) Genome editing tools in plants. Genes 8:399
Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52
Chapman JR, Taylor MRG, Boulton SJ (2012) Playing the end game: DNA double-strand break repair pathway choice. Mol Cell 47:497–510
Hefferin ML, Tomkinson AE (2005) Mechanism of DNA double-strand break repair by non-homologous end joining. DNA Repair 4:639–648
Lieber MR (2010) The mechanism of double-Strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181–211
Davis AJ, Chen DJ (2013) DNA double strand break repair via non-homologous end-joining. Transl Cancer Res 2:130–143
Zaboikin M, Zaboikina T, Freter C, Srinivasakumar N (2017) Non-homologous end joining and homology directed DNA repair frequency of double-stranded breaks introduced by genome editing reagents. PLoS One 12:1–36
Zhang F, Wen Y, Guo X (2014) CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet 23:40–46
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (New York, NY) 337:816–821
Lino CA, Harper JC, Carney JP, Timlin JA (2018) Delivering CRISPR: a review of the challenges and approaches. Drug Deliv 25:1234–1257
Bhowmik P, Ellison E, Polley B, Bollina V, Kulkarni M, Ghanbarnia K et al (2018) Targeted mutagenesis in wheat microspores using CRISPR/Cas9. Sci Rep 8:6502
Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123
Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaud J-B et al (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17:148
Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345
David H, David A, Mateille T (1982) Evaluation of parameters affecting the yield, viability and cell division of Pinus pinaster protoplasts. Physiol Plant 56:108–113
Roger D, David A, David HC (1996) Lmmobilization of flax protoplasts in agarose and alginate beads. Plant Physiol 112:1191–1199
Xie K, Yang Y (2013) RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant 6:1975–1983
Sauer NJ, Narváez-Vásquez J, Mozoruk J, Miller RB, Warburg ZJ, Woodward MJ et al (2016) Oligonucleotide-mediated genome editing provides precision and function to engineered nucleases and antibiotics in plants. Plant Physiol 170:1917–1928
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Adhikary, D., da Costa Ribeiro Quintans, I.L.A., Bhowmik, P.K. (2020). A Procedure to Design Guide RNA, Assemble Fragments, and Detect Mutation for Genome Editing in Flax. In: Islam, M.T., Bhowmik, P.K., Molla, K.A. (eds) CRISPR-Cas Methods . Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0616-2_11
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DOI: https://doi.org/10.1007/978-1-0716-0616-2_11
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