CRISPR-Cas9 Based Genome Editing in Wheat

• Main Authors: Clarke, M. (Author), Harwood, W.A (Author), Hayta, S. (Author), Smedley, M.A (Author)
• Format: Article
• Language: English
• Published: Blackwell Publishing Inc. 2021
• Subjects: amplicon; Article; Cas9; clustered regularly interspaced short palindromic repeat; Clustered Regularly Interspaced Short Palindromic Repeats; conserved sequence; controlled study; CRISPR; CRISPR associated endonuclease Cas9; CRISPR Cas system; CRISPR-Cas Systems; CRISPR-CAS9 system; crop production; double stranded DNA break; gene editing; Gene Editing; gene knockout; gene sequence; gene structure; genetic analysis; genetics; genome editing; Golden Gate assembly; guide RNA; hexaploidy; hybridization; knockout; nonhuman; plant breeding; Plant Breeding; plant genome; priority journal; Sanger sequencing; Triticum; Triticum aestivum; Triticum turgidum; wheat
• Online Access: View Fulltext in Publisher

 The development and application of high precision genome editing tools such as programmable nucleases are set to revolutionize crop breeding and are already having a major impact on fundamental science. Clustered regularly interspaced short palindromic repeats (CRISPR), and its CRISPR-associated protein (Cas), is a programmable RNA-guided nuclease enabling targeted site-specific double stranded breaks in DNA which, when incorrectly repaired, result in gene knockout. The two most widely cultivated wheat types are the tetraploid durum wheat (Triticum turgidum ssp. durum L.) and the hexaploid bread wheat (Triticum aestivum L.). Both species have large genomes, as a consequence of ancient hybridization events between ancestral progenitors. The highly conserved gene sequence and structure of homoeologs among subgenomes in wheat often permits their simultaneous targeting using CRISPR-Cas9 with single or paired single guide RNA (sgRNA). Since its first successful deployment in wheat, CRISPR-Cas9 technology has been applied to a wide array of gene targets of agronomical and scientific importance. The following protocols describe an experimentally derived strategy for implementing CRISRP-Cas9 genome editing, including sgRNA design, Golden Gate construct assembly, and screening analysis for genome edits. © 2021 The Authors. Basic Protocol 1: Selection of sgRNA target sequence for CRISPR-Cas9. Basic Protocol 2: Construct assembly using Golden Gate (MoClo) assembly. Basic Protocol 3: Screening for CRISPR-Cas9 genome edits. Alternate Protocol: BigDye Terminator reactions for screening of CRISPR-Cas9 genome edits. © 2021 The Authors.