Abstract
While public and political views on genetic modification (inserting “foreign” genes to elicit new traits) have resulted in limited exploitation of the technology in some parts of the world, the new era of genome editing (to edit existing genes to gain new traits/genetic variation) has the potential to change the biotech landscape. Genome editing offers a faster and simpler approach to gene knockout in both single and multiple genetic locations, within a single or small number of generations, in a way that has not been possible through alternative breeding methods. Here we describe an Agrobacterium-mediated delivery approach to deliver Cas9 and dual sgRNAs into 4-day-old cotyledonary petioles of Brassica oleracea. Mutations are detected in approximately 10% of primary transgenic plants and go on in subsequent T1 and T2 generations to segregate away from the T-DNA. This enables the recovery of non-transgenic, genome-edited plants carrying a variety of mutations at the target locus.
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References
Feng Z, Zhang B, Ding W, Liu X, Yang DL, Wei P, Zhu J-K (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Res 23(10):1229–1232
Li JF, Aach J, Norville JE, McCormack M, Zhang D, Bush J, Sheen J (2013) Multiplex and homologous recombination-mediated plant genome editing via guide RNA/Cas9. Nat Biotechnol 31(8):688–691
Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9:39
Shan Q, Wang Y, Li J, Zhang Y (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31(8):686–688
Lawrenson T, Shorinola O, Stacey N, Li C, Østergaard L, Patron N, Harwood W (2015) Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol 16:258
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 337:816–821
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Yang H, Wu J, Tang T, Liu K, Dai C (2017) CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus. Sci Rep 7(1):7489. https://doi.org/10.1038/s41598-017-07871-9
Yang Y, Zhu K, Li H, Han S, Meng Q, Khean S, Fan C, Xie K, Zhou Y (2018) Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development. Plant Biotechnol J 16(7):1322–1335. https://doi.org/10.1111/pbi.12872
Braatz J, Harloff H, Macher M, Stein N, Himmelbach A, Jung C (2017) CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus L.). Plant Physiol 174:935–942
Kirchner TW, Niehaus M, Debener T, Schenk MK, Herde M (2017) Efficient generation of mutations mediated by CRISPR/Cas9 in the hairy root transformation system of Brassica carinata. PLoS One 12(9):e0185429. https://doi.org/10.1371/journal.pone.0185429
Hundleby PAC, Irwin JAI (2015) Brassica oleracea and B. napus Agrobacterium Protocols. Springer Science+Business Media, New York, p 1223
Haeussler M, Schonig K, Eckert H, Eschstruth A, Mianne J, Renaud JB, Schneider-Maunoury S, Shkumatava A, Teboul L, Kent J, Joly JS, Concordet JP (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17:148
Doench JG, Fusi N, Sullender M, Hedge M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximise activity and minimise off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191
Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene editing in tomato in the first generation using clustered regularly interspersed short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166:1292–1297
Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V (2014) Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol 32:76–84
Acknowledgment
We acknowledge support from the Biotechnology and Biological Sciences Research Council (BBSRC) via grant [BB/N019466/1] and grant [BB/P013511/1] to the John Innes Centre. Further illustrative photographs of the transformation of B. oleracea DH1012 can be seen at www.jic.ac.uk/technologies/genomic-services/bract/. These transformation resources were developed as part of the Biotechnology Resources for Arable Crop Transformation (BRACT) facility, initially funded by Defra (UK) and now operating on a cost-recovery basis as a transformation resource for the research community.
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Lawrenson, T., Hundleby, P., Harwood, W. (2019). Creating Targeted Gene Knockouts in Brassica oleracea Using CRISPR/Cas9. In: Qi, Y. (eds) Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8991-1_12
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DOI: https://doi.org/10.1007/978-1-4939-8991-1_12
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