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Creating Targeted Gene Knockouts in Brassica oleracea Using CRISPR/Cas9

  • Tom Lawrenson
  • Penny Hundleby
  • Wendy Harwood
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)

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.

Key words

Brassica oleracea Vector CRISPR/Cas9 Knockout Mutant Gene edit Transgene free Genome editing 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.John Innes Centre, Norwich Research ParkNorwichUK

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