Design and assembly of crispr/cas9 reagents for gene knockout, targeted insertion, and replacement in wheat

Tomáš Čermák, Shaun J. Curtin

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Scopus citations

Abstract

Advances in cereal transformation along with the completion of the wheat genome sequence assembly have increased the demand for tools that perform targeted and specific modifications in this crop plant. This protocol demonstrates the construction of reagents using a comprehensive genome engineering kit to create single and multiple gene “knockouts,” site-specific chromosome deletions and gene replacement or “knockins” including the use of geminivirus replicons (GVRs). The reagents allow for both easy construction of simple genome engineering vectors, and “mix and match” swapping of components such as the Cas9, guide RNA and donor template cassettes for gene targeting. In addition, a web-based tool greatly streamlines vector selection, primer design, and vector construction.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages187-212
Number of pages26
DOIs
StatePublished - Jan 1 2017

Publication series

NameMethods in Molecular Biology
Volume1679
ISSN (Print)1064-3745

Keywords

  • CRISPR/Cas9
  • Chromosome deletion
  • GVRs
  • Gene insertion
  • Gene knockout
  • Gene replacement
  • Genome editing
  • Replicons
  • Wheat

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