CRISPR-Cas9 is a gene editing technology following the introduction of ZFN, TALENs and other gene editing technologies. Within a few years, CRISPR-Cas9 has become the most mainstream gene editing system in the world today.
CRISPR-Cas System
The CRISPR-Cas system is a natural immune system of prokaryotes, which consists of two parts, the CRISPR locus and the Cas gene (CRISPR-associated gene).
1. CRISPR is a sequence in the genome of prokaryotes, mainly composed of a leader sequence, a repeat sequence and a spacer sequence.
Leader sequence: rich in AT bases, located upstream of the CRISPR gene, considered to be the promoter of the CRISPR sequence.
Repeated sequence: about 20-50 bp in length and contains a 5-7 bp palindromic sequence. The transcript can form a hairpin structure and stabilize the overall secondary structure of the RNA.
Spacer sequence: is a foreign DNA sequence captured by bacteria. This is equivalent to the "blacklist" of the bacterial immune system. When these foreign genetic materials invade again, the CRISPR/Cas system will strike precisely.
2. The Cas gene is located near the CRISPR gene or scattered elsewhere in the genome, and the gene is named CRISPR-associated gene (Cas). The Cas protein encoded by the Cas gene is crucial in the defense process, and various types of Cas genes such as Cas1-Cas10 have been discovered.
According to the role of Cas proteins in the process of bacterial immune defense, CRISPR-Cas systems are currently divided into two categories.
The first category: their effectors for cutting exogenous nucleic acids are complexes formed by multiple Cas proteins, including type I, type III, and type IV.
The second category: their acting factors are relatively single Cas proteins, such as type II Cas9 protein and type V Cpf protein.
Currently, the most widely used CRISPR system is the type II CRISPR-Cas system, also known as the CRISPR-Cas9 system.
CRISPR-Cas9 Gene Editing Technology
CRISPR-Cas9 gene editing technology is to use artificially designed sgRNA (guide RNA) to identify the target genome sequence, and guide Cas9 protease to effectively cut the double strand of DNA, forming a double strand break, and the repair after the damage will cause gene knockout or knockin, etc., and finally achieve the purpose of modifying genomic DNA.
How does CRISPR-Cas9 Work?
The mechanism of action of CRISPR-Cas9 can be divided into three stages.
Phase 1: CRISPR's highly variable spacer captures foreign DNA
The highly variable spacer region of CRISPR captures a short DNA sequence of foreign invading phage or plasmid DNA, and integrates into the genome of the host bacterium. The integration position is between the two repeat sequences at the 5' end of CRRSPR. Thus, the arrangement of spacers in CRISPR loci from 5' to 3' also records the temporal order of invasion of foreign genetic material.
Phase 2: Expression of the CRIPSR locus
The CRISPR sequence is transcribed under the control of the leader region to produce pre-crRNA (precursor of crRNA), and tracrRNA (transactivation crRNA) complementary to the pre-crRNA sequence is also transcribed. The pre-crRNA forms a double-stranded RNA with the tracrRNA through complementary base pairing and assembles into a complex with the protein encoded by Cas9. It will select the corresponding "ID card number" (spacer sequence RNA) according to the type of intruder, and cut this "ID card" with the assistance of ribonuclease III (RNase III), and finally form a short crRNA (contains a single species of spacer RNA and a partial repeat region).
Phase 3: CRISPR/Cas system activity
The resulting complex of crRNA, Cas9, and tracrRNA acts like a guided missile, delivering precise strikes to the invader's DNA. This complex will scan the entire foreign DNA sequence and recognize the protospacer sequence that is complementary to the crRNA. At this time, the complex will localize to the PAM/protospacer region, and the DNA double strand will be unwound to form an R-Loop. The crRNA will hybridize to the complementary strand, while the other strand remains free.
Subsequently, the precise blunt-end cleavage site of the Cas9 protein is located 3 nucleotides upstream of the PAM, forming a blunt-end product. The HNH domain of the Cas9 protein is responsible for cutting the DNA strand complementary to crRNA, while the RuvC domain is responsible for cutting the other non-complementary DNA strand. Finally, under the action of Cas9, the DNA double strand breaks (DSB), the expression of foreign DNA is silenced, and the invaders are wiped out in one fell swoop.
Applications of CRISPR-Cas9
1. Knock-out
Cas9 can cut the target genome and form double-strand breaks in DNA. Normally, cells repair broken DNA using highly efficient non-homologous end joining (NHEJ). However, during the repair process, base insertion or deletion mismatches usually occur, resulting in frameshift mutations, (frameshift mutations: refers to changes in the reading frame of DNA molecules due to deletion or insertion of bases at a certain point, resulting in A series of downstream code changes, so that the gene encoding a certain peptide chain becomes another completely different peptide sequence.) The target gene loses its function, thereby achieving gene knockout. In order to improve the specificity of the CRISPR system, a domain of Cas9 can be mutated to form a Cas9 nickase nuclease that can only cut DNA single strands and cause DNA gaps. Therefore, if you want the effect of forming a double-strand break, you can design two sgRNA sequences to target the two complementary strands of DNA, so that the two sgRNAs can specifically bind to the target sequence to form a DNA break, and in the repair process through migration Coding mutations achieve gene knockout.
2. Knock-in
After a DNA double-strand break, if a DNA repair template enters the cell, the genome break will undergo homologous recombination repair (HDR) according to the repair template, thereby realizing gene knock-in. The repair template consists of the target gene to be imported and the homology sequences (homology arms) upstream and downstream of the target sequence. The length and position of the homology arms are determined by the size of the edited sequence. DNA repair templates can be linear/double-stranded deoxynucleotide strands, or double-stranded DNA plasmids. The HDR repair pattern occurs at a low rate in cells, usually less than 10%. In order to increase the success rate of gene knock-in, many scientists are currently working on improving the efficiency of HDR, synchronizing the edited cells to the most active cell division period of HDR, promoting the repair method by HDR, or using chemical methods to inhibit genes for NHEJ to improve HDR efficiency.
3. Gene Suppression or Gene Activation
The characteristic of Cas9 is that it can independently bind and cut the target gene, and the two domains RuvC- and HNH- of Cas9 are inactivated by point mutation, and the formed dCas9 can only bind the target gene under the mediation of sgRNA, without The function of cutting DNA. Therefore, binding dCas9 to the transcription initiation site of a gene can block the start of transcription, thereby inhibiting gene expression; binding dCas9 to the promoter region of a gene can also bind transcriptional repressors/activators, so that downstream target gene transcription is regulated. inhibit or activate. Therefore, the difference between dCas9 and Cas9 and Cas9 nickase is that the activation or inhibition caused by dCas9 is reversible and will not cause permanent changes to genomic DNA.
4. Multiplex Editing
Transfecting multiple sgRNA plasmids into cells can edit multiple genes at the same time, and can screen genome functions. Applications of multiple editing include the use of dual Cas9nickeses to improve the accuracy of gene knockout, large-scale genomic deletion, and simultaneous editing of different genes. Typically, 2 to 7 different sgRNAs can be constructed on one plasmid for multiple CRISPR gene editing.
5. Functional Genome Screening
Gene editing using CRISPR-Cas9 can generate a large number of genetically mutated cells, so using these mutated cells can confirm whether the phenotypic changes are caused by genes or genetic factors. The traditional method of genome screening is shRNA technology, but shRNA has its limitations: it has a high off-target effect and cannot inhibit all genes, resulting in false negative results. The genome screening function of the CRISRP-Cas9 system has the advantages of high specificity and irreversibility, and has been widely used in genome screening. At present, the genomic screening function of CRISPR is used to screen related genes that regulate phenotypes, such as genes that inhibit chemotherapy drugs or toxins, genes that affect tumor migration, and construct virus screening libraries for large-scale screening of potential genes.
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