Rice Genomics and Genetics 2024, Vol.15, No.1, 12-18 http://cropscipublisher.com/index.php/rgg 13 2 The Basic Principles of CRISPR-Cas9 Technology 2.1 Overview of multiple editing systems The multiple editing system is a further development of CRISPR-Cas9 technology, allowing for simultaneous editing of multiple genes or DNA loci to achieve more complex genetic improvements. The core of a multi editing system is to design multiple RNA guidance sequences, each sequence used to guide Cas9 protein to different target sites. These RNA guidance sequences typically complement and pair with different target genes or DNA sequences. Therefore, multiple editing systems require scientists to carefully design multiple different RNA guidance sequences to meet the needs of editing multiple loci. In a multi editing system, each designed RNA guide sequence typically binds to a Cas9 protein. This means that multiple Cas9 proteins simultaneously act on different target sites. These Cas9 proteins can be the same or different variants of Cas9, depending on the research needs (Figure 1) (Xu et al., 2022). Figure 1 Mutants of Cas proteins and different species of Cas proteins can to some extent expand the targeting range of CRISPR tools (Adopted from Xu et al., 2022) By using a multiple editing system, scientists can simultaneously edit multiple DNA sites. This allows them to achieve more complex genetic improvements, such as multi gene regulation, optimization of complex metabolic pathways, and more functional enhancements. Multi editing systems have wide applications in fields such as agriculture, medicine, biological research, and biotechnology. For example, it can be used to develop multi resistant crop varieties, treat polygenic genetic diseases, or achieve regulation of multiple cytokines. Multiple editing systems provide more possibilities for solving complex biological problems (Xin et al., 2018). Despite the enormous potential of multiple editing systems, there are also challenges and limitations. This includes complex RNA guided sequence design, interference between multiple Cas9 proteins, and potential safety and ethical issues. In addition, editing multiple loci may lead to unwanted side effects. The introduction of multiple editing systems has expanded the application scope of CRISPR-Cas9 technology, enabling scientists to perform gene editing and genetic improvement more comprehensively. However, it requires careful planning and precise experimental design to ensure the desired editing effect and minimize unwanted impacts. 2.2 Mechanism of Cas9 protein and RNA guided action The core mechanism of CRISPR-Cas9 technology lies in the synergistic effect guided by Cas9 protein and RNA to achieve precise gene editing. Cas9 is an endonuclease of the CRISPR-Cas9 system, responsible for cleaving target DNA. The Cas9 protein has two main functional domains: RuvC and HNH. These two functional domains work together to bind Cas9 protein to target DNA and cause DNA double strand breaks. In the CRISPR-Cas9 system, the RNA guide sequence is designed by scientists to complement and pair with the DNA sequence of the target gene. This RNA guidance sequence is merged into the CRISPR-Cas9 system to guide Cas9 protein binding to specific DNA sites. Accurately designed RNA guidance sequences are key to achieving specific gene editing. The Cas9 protein forms a Cas9 RNA complex through complementary pairing with RNA guiding sequences. This complex has high specificity, allowing it to recognize and bind to the target DNA. Once the Cas9 RNA complex binds to the target DNA, it forms a DNA-RNA double helix structure that locates the Cas9 protein at a specific location of the target gene.
RkJQdWJsaXNoZXIy MjQ4ODYzNA==