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 language | English (US) |
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Title of host publication | Methods in Molecular Biology |
Publisher | Humana Press Inc. |
Pages | 187-212 |
Number of pages | 26 |
DOIs | |
State | Published - 2017 |
Publication series
Name | Methods in Molecular Biology |
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Volume | 1679 |
ISSN (Print) | 1064-3745 |
Bibliographical note
Publisher Copyright:© 2017, Springer Science+Business Media LLC.
Keywords
- CRISPR/Cas9
- Chromosome deletion
- GVRs
- Gene insertion
- Gene knockout
- Gene replacement
- Genome editing
- Replicons
- Wheat