Figure 1. Schematic diagram of gene editing ^[1]

Research history

In the early 1980s, the success of embryonic stem cell isolation and in vitro culture laid the technical foundation for gene knockout. In 1985, the existence of homologous recombination (HR) in mammalian cells, which was first confirmed, laid the theoretical foundation for gene knockout ^[2] . In order to edit genes, traditional homologous recombination techniques targeting specific alleles are used. However, this method has the disadvantages of low efficiency and high labor cost in the past, which seriously restricts basic research and clinical application. Scientists need to explore more concise and efficient gene editing techniques.

In order to realize the use of simple methods to disable specific genes, scientists have developed RNA interference (RNAi) technology, which is expected to make it easier to study the gene function of mammalian cells. It has the advantages of simple operation and obvious effects. However, RNAi cannot act on all genes and certain cell types (such as neurons), and it has the disadvantages of positional effects, temporary and incomplete knockouts ^[3] .

With the advancement of various gene editing technologies, such as: zinc finger nuclease (ZFN) ^[4] , transcription activator-like effector nucleases (TALENs) ^[5] and Clusters of regularly spaced short palindromic repeats (CRISPR/CRISPR-associated (Cas) 9 system, CRISPR/Cas9) ^[6] and other technical changes, the field of gene editing has also occurred Revolutionary change.

Today, Xiaobian will bring you back to the world of CRISPR/Cas9 technology and RNA interference . Let’s review history together~

Figure 2. http:// ^[7]

CRISPR/Cas9 gene knockout

Knockout refers to the use of genetic engineering techniques to change the genetics of a specific known gene sequence, to disable its function, and further affect the organism, and then to speculate on the biology of the gene. Features. CRISPR/Cas9 is the fastest-growing new gene knockout technology in recent years. It is widely distributed in many prokaryotic genomes. The domain-type CRISPR/Cas9 system can rely on the Cas9 endonuclease family to target the cleavage of foreign DNA. After transcription, each crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) bind together and form a complex with Cas9 nuclease ^[3] . The Cas9 nuclease, under the guidance of crRNA, recognizes a conserved spacer motif (PAM) and binds to DNA to cleave DNA. This technology is simple, fast, low-cost, and has low off-target effects. It has become a powerful weapon in the field of gene function research, greatly speeding up the screening and verification of drug targets and the development of new drugs.

Figure 3. Schematic diagram of the work of CRISPR/Cas9 technology ^[8]

RNA interference

RNA interference refers to the phenomenon that small double-stranded RNA can specifically degrade or inhibit homologous mRNA expression, thereby inhibiting or shutting down the expression of specific genes, mainly through short interfering RNA (siRNA) and short hairpin RNA. (short hairpin RNA, shRNA) to regulate gene expression ^[9] . Under the action of Dicer, dsRNA can produce a series of 21~22 nt siRNAs, which are melted into the sense and antisense strands by intracellular RNA helicase, followed by antisense siRNA and some enzymes in vivo. (including endonuclease, exonuclease, helicase, etc.) bind to form an RNA-induced silencing complex (RISC). The nuclease-functional RISC specifically binds to the mRNA homologous region expressed by the exogenous gene, and cleaves the mRNA at the binding site. The cleavage mRNA after cleavage is then degraded, thereby inducing host cell degradation reaction to these mRNAs, blocking the expression of endogenous genes, inactivating the gene, and finally achieving the effect of gene silencing ^[3] .

Figure 4. Schematic of RNA interference ^[10]

Comparison of CRISPR/Cas9 gene knockout and RNA interference

For now, the efficiency of CRISPR/Cas9 technology is already very high, and it is basically possible to obtain knockout mice after a single target process, while traditional homologous recombination-based techniques take longer. Based on the advantages of low cost, simple production, fast and efficient, CRISPR/Cas9 gene knockout technology has quickly become popular in laboratories around the world and has become an effective tool in scientific research and medical fields. It is considered to be the most active cell. Effectively and conveniently "edit" any gene; as a novel targeted gene editing technology, CRISPR/Cas9 technology has been widely used in many species such as zebrafish, mice and rats. The technology does not have the limitations of animal species, and it has also made breakthroughs in plant genetic modification, such as soybean, tobacco, rice and wheat.

The advantage of CRISPR technology is that it permanently alters the sequence of genomic DNA, but RNAi technology has its unique advantages in gene silencing. Specifically, RNAi is first safer and less costly without the introduction of additional protein factors. Secondly, RNAi is a reversible gene silencing technology at the post-transcriptional level, either by the addition of siRNA or by the use of small molecule compounds to regulate siRNA. The activity of the inducible promoter on the expression vector can achieve the silencing and opening of the target gene; finally, RNAi regulates the expression of the gene at the post-transcriptional level, so the siRNA design is only required to refer to the transcriptome data ^[11] .

application

1. CRISPR/Cas9 gene knockout

CRISPR/Cas9 technology in the functional gene screening process of drug development, only need to import a sgRNAs library into the cell, and then through the evaluation of the relevant phenotype, can achieve large-scale fixed-point editing and screening of the genome, and then reveal the physiological physiology of the gene. Function to provide reliable targets for new drug development.

The CRISPR/Cas9 technology is not limited to gene knockouts, including large-segment knocking, point mutations, and humanized replacement strategies, which have already been used in animal model construction, greatly reducing the time and expense of building animal models. Moreover, it has been widely used in the construction of various gene editing models (mainly including tumor and stem cell lines). In addition, the application of this technology to human gene therapy is also in the process of rapid development, which will bring great help to the treatment of human diseases ^[12] .

2, RNA interference

1 gene function research

Because of its good specificity and effective interference activity, RNAi can silence specific genes, lose their function or reduce expression, so it can be used as a powerful research tool for functional genomics ^[9] . Studies have shown that siRNA can inhibit the expression of specific genes in mammals, and the time to inhibit gene expression can be controlled at any stage of development, producing similar knockout effects.

2 treatment of viral diseases

Studies have shown that by synthesizing a siRNA targeting a specific viral gene and introducing it into the virus-infected cells, it is possible to effectively inhibit the replication of the virus and block the infection of the virus by the virus. Moreover, it is valuable that siRNA can exert an inhibitory effect in the early stage of viral infection. Thus, siRNA can be used to treat viral diseases.

3 treatment of tumor disease

Tumors are the result of gene network regulation of multiple gene interactions. The blocking of a single oncogene induced by traditional techniques cannot completely inhibit or reverse the growth of tumors, and RNAi can utilize multiple genes of the same gene family to have a homology. A highly conserved sequence, the siRNA molecule targeting this sequence can be injected with only one siRNA to produce multiple genes simultaneously, or multiple siRNAs can be injected simultaneously. The genes are simultaneously eliminated.

to sum up

In summary, in gene editing technology, gene knockout technology and RNAi technology have their own advantages, have been actively applied to the study of some biological problems, including some human diseases, greatly satisfying the purpose of researchers to operate different types of genomes, Not only as a tool for gene editing, but also for gene expression regulation. This is like "the sword and the dragon, commanding the world; relying on the sky, who is fighting for the front."

Today's dry goods are here for the time being. Are the babies full of ink? Cough, if you are interested, be sure to pay attention to the small series at any time. You will not miss what you want. Let's meet again~ Quietly come, and walk gently, don't take any regrets.

Reference material

[1]https://

[2]Smithies O, Gregg RG, Boggs SS, et al. Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination [J]. Nature, 1985, 317(6034): 230-234.

[3]LI Wei, ZHAO Hongye, CUI Yong, WEI Hongjiang, LI Meizhang. Research progress of gene editing technology[J].Life Science Research,2017,21(3)

[4] Hauschild-Quintern J, Petersen B, Cost GJ, et al. Gene knockout and knockin by zinc-finger nucleases: current status and perspectives[J]. Cellular and Molecular Life Sciences, 2013, 70(16): 2969- 2983.

[5]Cermak T, Doyle EL, Christian M, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting [J]. Nucleic Acids Research, 2011, 39(12): e82.

[6]Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems [J]. Science, 2013, 339(6121): 819-823

[7]http://

[8]https://

[9] Wilson RC, Doudna J A. Molecular mechanisms of RNA interference [J]. Annual Review of Biophysics, 2013, 42: 217-239

[10]http://blog.sina.com.cn/s/blog_445dac3b0102x8kb.html

[11] Shang Renfu, Wu Ligang. Mechanism of RNA interference and its application[J]. Life Science, 2016, 28(5)

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