Bioscience Methods 2024, Vol.15, No.6, 315-326 http://bioscipublisher.com/index.php/bm 319 effectiveness of CRISPR/Cas9 in addressing various abiotic stresses, paving the way for the development of resilient wheat varieties capable of thriving in challenging environments (Figure 2) (Biswas et al., 2021; Wang et al., 2022). Figure 2 The methodology of major CRISPR/Cas systems (Adopted from Wang et al., 2022) Image caption: (A) CRISPR/Cas9 induces double-stranded breaks (DSBs) in DNA strands. (B) CRISPR/Cas12a cleaves the target DNA and introduces DSBs. (C) CRISPR/Cas methods can achieve different research goals: (a-c) are results of non-homologous end-joining NHEJ, and (d,e) are results of the homology-directed repair HDR repair pathways using a donor DNA template. (D–F) Base editing tools mainly include Cytidine Base Editor (CBE), Adenine Base Editor (ABE), and Prime Editor (PE). (D) CBE converts C-G base pairs to T-A base pairs at the target site. (E) ABE converts A-T base pairs to G-C base pairs at the target site. (F) PE is a new base editing system, which enables precise sequence substitution, insertion, and deletion. PE mainly consists of a Cas9 nickase (nCas9), an engineered reverse transcriptase (RT), and pegRNA. PegRNA includes PBS (Primer Binding Site) sequence and RT Template. (G) CRISPR/Cas13 consists of a Cas13, a crRNA, and a target RNA. Cas13:crRNA complexes bind target RNA and cleave the target RNA. (H) CRISPR transcriptional activation (CRISPRa) consists of a nuclease-deficient Cas9 (dCas9) and transcription activation domain (TAD). CRISPRa activates the transcription of single or multiple target genes (Adopted from Wang et al., 2022) 5 Improvement of Wheat Yield and Quality 5.1 Key traits related to yield (e.g., grain size, grain weight) The application of CRISPR/Cas9 technology in wheat has shown significant potential in enhancing key yield-related traits such as grain size and grain weight. By targeting specific genes that regulate these traits, researchers have been able to create wheat varieties with improved yield characteristics. For instance, the manipulation of genes involved in the CLAVATA-WUSCHEL pathway, which controls meristem size, has been shown to increase grain yield in maize, suggesting similar potential in wheat (Liu et al., 2021b). Additionally, the editing of genes related to nitrogen use efficiency, such as the ARE1 ortholog, has resulted in wheat varieties with increased grain yield under nitrogen-limiting conditions (Zhang et al., 2021a). Moreover, CRISPR/Cas9 has enabled the precise editing of regulatory genes that control grain size and weight, leading to the development of wheat varieties with enhanced yield potential. This technology allows for the creation of new allelic variations in a much faster and more precise manner compared to traditional breeding methods (Zhang et al., 2021). The ability to target multiple genes simultaneously further enhances the potential for yield improvement, making CRISPR/Cas9 a powerful tool in wheat genetic improvement (Arora and Narula, 2017).
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