Bioscience Methods 2024, Vol.15, No.6, 315-326 http://bioscipublisher.com/index.php/bm 316 2 Overview of CRISPR/Cas9 Technology 2.1 Basic working principles of CRISPR/Cas9 The CRISPR/Cas9 system, derived from the adaptive immune system of bacteria, has revolutionized genome editing due to its simplicity and efficiency. The system consists of two main components: the CRISPR sequences and the Cas9 protein. CRISPR sequences are short, repetitive DNA sequences found in the genomes of bacteria and archaea, which, when transcribed, form CRISPR RNA (crRNA). These crRNAs guide the Cas9 protein to specific DNA sequences in the genome, where Cas9 introduces double-strand breaks (DSBs) (Arora and Narula, 2017; Bao et al., 2019). The DSBs are then repaired by the cell's natural repair mechanisms, leading to targeted mutations. The Cas9 protein is an endonuclease that can be programmed to cut DNA at specific sites by using a single-guide RNA (sgRNA). The sgRNA is a synthetic fusion of crRNA and a trans-activating crRNA (tracrRNA), which simplifies the system by combining the targeting and binding functions into a single molecule (Bao et al., 2019; Montecillo et al., 2020). This RNA-guided mechanism allows for precise targeting of genomic sequences, making CRISPR/Cas9 a versatile tool for genetic engineering. 2.2 The role of sgRNA design in targeted editing The design of the sgRNA is crucial for the specificity and efficiency of the CRISPR/Cas9 system. The sgRNA contains a 20-nucleotide sequence that is complementary to the target DNA sequence, guiding the Cas9 protein to the correct location in the genome (Shan et al., 2014; Doench et al., 2015). The precision of this targeting is essential to minimize off-target effects, which can lead to unintended mutations in the genome. Various computational tools and design rules have been developed to optimize sgRNA sequences, enhancing their on-target activity while reducing off-target effects (Doench et al., 2015; Manghwar et al., 2020). Effective sgRNA design involves selecting target sites that are unique within the genome and ensuring that the sgRNA has a high binding affinity for the target sequence. Additionally, the presence of a protospacer adjacent motif (PAM) sequence, typically NGG for the commonly used Streptococcus pyogenes Cas9, is necessary for Cas9 binding and cleavage (Montecillo et al., 2020; Zhang et al., 2023). Advances in deep learning and other computational methods have further improved the accuracy of sgRNA design, enabling more efficient and precise genome editing (Zhang et al., 2023). 2.3 Advantages and limitations of the CRISPR/Cas9 system in plant gene editing The CRISPR/Cas9 system offers several advantages for plant gene editing. Its simplicity and versatility allow for the rapid generation of targeted mutations, facilitating the study of gene function and the development of new plant traits (Kim et al., 2017; Bao et al., 2019). The system can be used to create knockouts, insertions, and precise modifications, making it a powerful tool for crop improvement. Additionally, the ability to multiplex, or target multiple genes simultaneously, further enhances its utility in complex plant genomes (Arora and Narula, 2017; Montecillo et al., 2020). However, the CRISPR/Cas9 system also has limitations. One of the main challenges is the potential for off-target effects, which can lead to unintended genetic changes. This is particularly problematic in plants with complex and polyploid genomes, such as wheat, where distinguishing between homologous sequences can be difficult (Kim et al., 2017; Cui et al., 2019). Moreover, the efficiency of CRISPR/Cas9-mediated editing can vary depending on the target site and the delivery method used. Despite these challenges, ongoing research and technological advancements continue to improve the precision and efficiency of the CRISPR/Cas9 system in plant gene editing (Jiang et al., 2013; Cui et al., 2019). 3 Wheat Disease Resistance Improvement 3.1 The threat of major wheat diseases to yield Wheat is a staple crop globally, but its production is significantly threatened by various diseases, notably stripe rust (Puccinia striiformis f. sp. tritici) and leaf rust (Puccinia triticina). These diseases can cause substantial yield losses, with stripe rust alone capable of reducing wheat yields by up to 70% in severe epidemic years (Yuan et al.,
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