title: RNA-guided genome editing for target gene mutations in wheat.
abstract: The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas-mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3' end of the target site abolished the cleavage activity completely. The mismatches at the 5' end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3' end. This approach provides a powerful method for genome engineering in plants.
authors.affiliation: National Agri-Food Biotechnology Institute, Department of Biotechnology, Government of India, Mohali, Punjab, India 160071.
authors.forename: Santosh Kumar
authors.initials: SK
authors.lastname: Upadhyay
authors.forename: Jitesh
authors.initials: J
authors.lastname: Kumar
authors.forename: Anshu
authors.initials: A
authors.lastname: Alok
authors.forename: Rakesh
authors.initials: R
authors.lastname: Tuli
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018506:Gene Expression Regulation, Plant; D018390:Gene Targeting / Q000379:methods*; D018745:Genome, Plant; D050562:Inositol Oxygenase / Q000235:genetics; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics; D018515:Plant Leaves / Q000235:genetics; D017393:RNA Editing; D017384:Sequence Deletion; D014026:Nicotiana / Q000235:genetics; D014908:Triticum / Q000235:genetics*; D058727:RNA, Small Untranslated
chemical_list: D010088:Oxidoreductases; D050562:Inositol Oxygenase; C022398:phytoene dehydrogenase; D058727:RNA, Small Untranslated
keywords: CRISPR; Cas; cgRNA; genome editing; indel; wheat genome editing