1 The CRISPR/Cas9 system and its evolution

In 1987, Ishino et al. discovered spaced repeats in the genome of E. coli. It was not until 2002 that Jansen et al. named it "Clustered Regularly Interspaced Short Palindromic Repeats, abbreviated as CRISPR".CRISPR is widely found in bacteria and archaea, and has important roles such as antiviral infection, DNA repair and modification, etc. It was not until 2012 that Doudna and Charpentier demonstrated that CRISPR/Cas9 could be used in the genome of E. coli. It was not until 2012 that Doudna and Charpentier proved that CRISPR has the ability of gene editing. In the same year, Sternberg et al. found that Cas9 protein could accomplish target sequence editing. A year later, Cong et al. applied CRISPR/Cas9 to mammalian genome editing for the first time.

The CRISPR/Cas9 system consists of three main components: crRNA, tracrRNA and Cas9 protein. Based on the principle of base complementary pairing, crRNA and tracrRNA can form a crRNA-tracrRNA structure, embedded in a small guide RNA (sgRNA), which guides the Cas9 protein to make double-stranded cuts in the target DNA sequence. Before target DNA cleavage, Cas9 protein, guided by sgRNA, recognizes the neighboring motifs of the original spacer sequence and cleaves the target DNA sequence upstream of the spacer sequence.After DNA double-strand breakage, the repair mechanism mainly relies on non-homologous end joining (NHEJ) and homology- directed repair (HDR), and the repair mechanism is mainly based on homology and homology-mediated repair. NHEJ is a random repair mechanism, which can cause DNA sequence shift mutation by insertion or deletion to achieve gene knockout or genome component failure, while HDR is a precise repair mechanism, which relies on importing exogenous DNA template and homologous recombination at the break to achieve targeted insertion, deletion, mutation, etc. CRISPR/Cas9 system has been used in the gene editing process, which is the most effective way of repairing DNA. The CRISPR/Cas9 system is a milestone in the history of gene editing, and has been widely used in medicine, agriculture, life science and other disciplines. It has been widely used in medicine, agriculture, life sciences and other disciplines. It is of great significance that researchers can utilize the CRISPR/Cas9 system to study disease models, develop therapeutic drugs, prepare transgenic animals, and study gene functions.

2 Overview of EVs

CRISPR/Cas9 system can perform gene editing rapidly and efficiently, and has an important role in disease modeling and disease treatment, etc. However, there is still a lack of safe and effective delivery vectors, which limits the clinical application of CRISPR/Cas9 system. Currently, EVs are one of the most promising delivery vectors, which are mainly classified into exosomes, microvesicles and apoptotic vesicles according to their sizes and origins, among which apoptotic vesicles and microvesicles can be produced by direct germination through the cell membrane, whereas exosomes are the intraluminal vesicles released after the fusion of multivesicular bodies and lysosomes, or vesicles released after the fusion with the cell membrane. As a natural nanoscale vector, EVs have significant advantages over traditional viral and non-viral vectors, such as excellent biocompatibility and low immunogenicity. In addition, EVs can carry biomolecules between cells and cross biological barriers such as blood-brain barrier, placental barrier and intestinal barrier. Therefore, EVs are expected to be the most promising delivery vehicles. In particular, EVs can deliver ribonucleoprotein (RNP) complexes, which are highly desirable delivery substances that are complexes of specific proteins and specific RNAs, including sgRNAs complexed with Cas9 proteins in vitro. By minimizing the potential risk of DNA sequence integration and RNA degradation, RNPs can improve the efficiency of gene editing and play an important role in the treatment of hereditary or infectious diseases. Therefore, we can foresee the potential value of EVs as vectors for delivering the advantages of the CRISPR/Cas9 system in the future.

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