International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 8-15 http://medscipublisher.com/index.php/ijmms 9 1 The Basic Principles and Technological Innovations of CRISPR-Cas9 Technology 1.1 Discovery and evolution of CRISPR-Cas9 technology CRISPR-Cas9 technology was initially discovered in bacterial genomes (Zhu et al., 2019). In the 1990s, researchers identified a class of repetitive sequences called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), the function of which remained unknown for a long time. Subsequent studies revealed that CRISPR sequences were highly similar to viral sequences in bacteriophage or plasmid genomes. Further research has found that the CRISPR immune system is a natural defense mechanism of bacteria against viral invasions. The CRISPR-Cas system consists of CRISPR sequences and associated Cas proteins, with Cas proteins being crucial factors capable of recognizing and cleaving foreign DNA. In 2012, Emmanuelle Charpentier, along with a research team from Stanford University, discovered a type II CRISPR-Cas system, which includes a protein called Cas9. The Cas9 protein exhibits highly effective gene editing capabilities. The CRISPR-Cas9 technology has garnered significant attention and widespread application due to its simplified design, convenient operation, precise gene editing, broad applicability, and relatively low cost and time consumption. The further development and improvement of CRISPR-Cas9 technology are ongoing. Scientists through in-depth studies of the structure and function of the Cas9 protein, have developed various Cas9 variants, such as nuclease-dead Cas9 (dCas9) and high-fidelity Cas9. Additionally, the introduction of modified single-guide RNA (sgRNA) and chemically modified primers has further enhanced the efficiency and precision of gene editing. 1.2 Basic principles and working mechanism of CRISPR-Cas9 technology CRISPR sequences consist of a set of repetitive DNA sequences and interspersed viral or plasmid DNA segments derived from bacterial or archaeal genomes. These sequences serve as the immune system of bacteria, used to store previously encountered foreign DNA segments to defend against future viral attacks. Through a series of DNA modifications and RNA processing steps, the CRISPR sequences are transcribed into a type of RNA molecule called CRISPR RNA (crRNA). In the CRISPR-Cas9 system, scientists design and construct an artificially synthesized RNA molecule called single-guide RNA (sgRNA). It is formed by the fusion of crRNA and a transcribed RNA segment known as tracrRNA. This sgRNA can bind to the Cas9 protein, forming a complex with the ability to guide Cas9 to recognize and bind to specific DNA sequences (Wang et al., 2019). The Cas9 protein is a central component of the CRISPR-Cas9 system, possessing DNA binding and cleavage functions. Once the sgRNA binds to the Cas9 protein, the complex can recognize a specific region of the target DNA sequence that pairs with the sgRNA. Upon binding to the specific DNA sequence, the Cas9 protein induces a double-strand break, resulting in alterations to the genomic DNA sequence. Following the formation of the double-strand break, the cell initiates its own DNA repair mechanism, attempting to mend the break. Through different repair mechanisms, such as non-homologous end joining or homologous recombination, precise editing of the target gene can occur, leading to insertions, deletions, or replacements in the genome (Figure 1). 1.3 Comparison between CRISPR-Cas9 and traditional gene editing technologies GCRISPR-Cas9 exhibits significant advantages compared to traditional gene editing technologies. The CRISPR-Cas9 technology allows for highly precise gene editing. Different from traditional methods, Cas9 protein can accurately locate and cleave the target DNA sequence by designing appropriate sgRNA. CRISPR-Cas9 technology is highly flexible, as changing the target gene simply involves replacing the sgRNA sequence, whereas traditional methods often require multiple optimizations. Additionally, CRISPR-Cas9 technology is cost-effective and time-saving, utilizing sgRNA and Cas9 protein as the main tools without the need for expensive recombinant enzymes and reagents. Lastly, CRISPR-Cas9 technology is applicable to a variety of organisms, including cells and whole organisms, while traditional methods are typically limited to specific cell types or species (Cao et al., 2020).
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