The full name of the CRISPR/Cas system is clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins. This system is an acquired immune system derived from bacteria and has been developed by researchers as a tool for targeted editing at specific DNA sites. The CRISPR/Cas9 system consists of Cas9 endonuclease and sgRNA: Cas9 endonuclease protein: contains RuvC and HNH-like nuclease domains, has the function of cutting double-stranded DNA, can recognize the PAM motif (5'-NGG), and cuts double-stranded DNA at the third base upstream of the PAM motif. sgRNA: a complex composed of complementary pairing of tracrRNA and crRNA partial sequences; tracrRNA is responsible for binding to Cas9 protein, while crRNA carries foreign nucleic acid recognition sequence (spacer). In gene editing, in order to facilitate experimental design and improve the stability of gRNA, tracrRNA and crRNA are fused into one RNA, called sgRNA.
CRISPR/Cas9 delivery tool
In practical applications, plasmids, Cas proteins and sgRNA, mRNA and sgRNA, circular RNA (circRNA) and sgRNA can be delivered to cells or bodies through cell transfection, adeno-associated virus (AAV), lentivirus and other viral packaging, exosome packaging, LNPs encapsulation, etc., so as to express the CRISPR/Cas9 system in vivo or intracellularly to achieve the purpose of gene editing.
Each form has its own advantages and disadvantages, and the expression form of circRNA is the best prospect and development potential.
1. Plasmid
Advantages: relatively stable, low cost, easy to manufacture;
Disadvantages: low transfection and expression efficiency; high risk of integration into the genome; large Cas protein, resulting in a large number of plasmid bases, which increases the difficulty of virus packaging.
2. Cas protein and sgRNA complex
Advantages: The method is relatively direct and effective quickly;
Disadvantages: The complex degrades quickly; The protein is large and the charge characteristics of the RNPs complex make the delivery efficiency low; The purer active Cas protein takes a long time and is costly, and there is also the problem of bacterial endotoxin contamination.
3. mRNA and sgRNA
Advantages: It is easy to obtain and can be obtained by in vitro transcription; It is more effective than plasmids; The probability of off-target is lower; The immunogenicity is lower; The intracellular residence time is short and the safety is higher;
Disadvantages: mRNA is unstable and easily degraded.
4. Circular RNA (circRNA) and sgRNA
Advantages: It is more effective than plasmids, has a lower off-target efficiency, is easier to obtain, is safer, and can be prepared in vitro (with all the advantages of linear mRNA); Compared with linear mRNA, the circular structure of circRNA makes it more stable and has a longer half-life. The immunogenicity of linear RNA may interfere with protein production and trigger an immune response to eliminate edited cells. Therefore, circRNA with lower immunogenicity is a more suitable medium for gene editing elements. Therefore, circRNA has important application prospects in gene editing.
Disadvantages: Currently, the application development of RNA therapy is mainly focused on linear mRNA, and the development research of circRNA application is still relatively small, and it is still at the conceptual level. Researchers still have reservations about the translation ability and in vitro preparation of circRNA.
Advantages of CRISPR/Cas9
1. Efficiency and accuracy: CRISPR/Cas9 technology can perform gene editing in an efficient and accurate manner, especially with extremely high accuracy in identifying and targeting specific sites of genes.
2. Multiple gene editing: Multiple gene sites can be edited at the same time, which is very important in functional genomics research.
3. Wide adaptability: CRISPR/Cas9 technology can be applied to a variety of biological systems, including bacteria, yeast, plants, animals and human cells.
4. Simple and easy to operate: Compared with other gene editing technologies (such as ZFN and TALEN), CRISPR/Cas9 is simpler to operate, takes less time and costs less.
Applications of CRISPR/Cas9
1. Gene knockout: Through the NHEJ repair mechanism, CRISPR/Cas9 can introduce small insertion or deletion mutations, thereby destroying the function of genes and achieving the purpose of knocking out specific genes in cells or organisms. This is very useful for studying gene function and establishing disease models.
2. Gene editing and gene repair: Through the HDR repair mechanism, researchers can use CRISPR/Cas9 to introduce specific DNA sequences to repair mutant genes or insert new genes. This application has great potential in the treatment of genetic diseases.
3. Gene activation or repression (CRISPRa/CRISPRi): Use an inactivated Cas9 (dCas9) protein that cannot cut DNA but can still target specific sequences. By binding to transcription activators or repressors, dCas9 can be used to upregulate or downregulate the expression of specific genes without changing the gene sequence.
4. Establishment of disease models: Through gene knockout or knock-in technology, CRISPR/Cas9 can be used to create animal models with specific gene mutations for studying the mechanisms of human diseases, especially cancer, neurodegenerative diseases and genetic diseases.
5. Large-scale genome screening: Through genome-wide gene knockout screening with CRISPR/Cas9, key genes related to a certain biological process or disease can be discovered. This technology is particularly important in drug target discovery and functional genomics research.
6. Gene therapy: Infectious diseases can be treated through targeted gene editing of viruses; tumor diseases can be treated by constructing tumor models, targeted knockout of oncogene sites, restoring tumor suppressor gene activity, reducing tumor cell resistance, activating tumor immunity, etc.; genetic diseases can be treated by cutting DNA double strands at or near the mutation site and inducing different types of insertions at the same time, resulting in gene deletion, mutation, knock-in, inversion, etc.; gene editing can be used to solve problems in xenotransplantation.
7. Molecular diagnosis: Using the highly specific recognition and binding characteristics of Cas9 for double-stranded DNA (dsDNA), combined with PCR technology, it can achieve nucleic acid molecule detection with higher sensitivity, specificity and universality.
8. Antibody production: Editing animal immune molecule genes into human or xenogeneic genes will solve the problem of xenogeneic antibody immune rejection.
9. Cultivating new varieties: Through the directional transformation of organisms, new varieties of crops, livestock and poultry, aquatic products, etc. can be cultivated.
References
https://en.wikipedia.org/wiki/CRISPR_gene_editing