Three Genome Editing Techniques: CRISPR, TALEN
Gene editing technology refers to the ability to allow humans to "edit" target genes to achieve the knockout and addition of specific DNA fragments. Since the advent of CRISPR/Cas9 technology, it has unparalleled advantages in other gene editing technologies. After continuous improvement of the technology, it is considered to be the most effective and convenient to "edit" any gene in living cells.
The methods of gene editing technology include:
- Gene knockout: make the function of a gene lost. During the repair process, DNA insertion or deletion markers are often generated to cause mutations in the transfer code, thereby achieving gene knockout;
- The introduction of specific mutations: introduce a specific mutation into the genome;
- Site-specific transgene: the same principle as the introduction of specific mutations, add a transgene in the middle of the homology template.
Three major techniques for genome editing: CRISPR, TALEN and ZFN.
- Zinc Finger Nuclease (ZFN)
The first genome editing strategy using custom DNA endonuclease is zinc finger nuclease (ZFN for short).
Zinc finger proteins are transcription factors; each finger module recognizes a sequence of 3-4 bases, mix and match these modules, and researchers can more or less target any sequence they want.
ZFN is a heterodimer, where each subunit contains a zinc finger domain and a FokI endonuclease domain. The FokI domain must be dimerized to be active, ensuring that there must be two adjacent DNA binding events to achieve double-strand breaks, thereby increasing target specificity.
Cleavage events have enabled most genome editing techniques. After the double strand breaks, the cell tries to repair it. The simplest method is non-homologous end joining (NHEJ), in which cells essentially flatten the ends of the broken DNA and draw them closer to each other, which often results in frameshifts. Another method is homologous directed repair (HDR). The cell tries to use the corresponding DNA sequence on another chromosome as a template to repair the break. By providing their own templates, users can force the system to inadvertently insert the required sequence.
- Transcription activator-like effector nuclease (TALEN)
Although zinc finger nucleases are not bad, they are more expensive to make and more cumbersome. Later, a similar but more flexible system appeared—TALEN.
TALENs have the function of destroying or changing specific genes of zebrafish, Xenopus and other animals. TALENs are a protein that can selectively cut specific DNA sequences and can selectively repair specific genes.
Transcription activator-like effector nuclease (TALEN) is a dimeric transcription factor/nuclease consisting of 33 to 35 amino acid modules, each of which targets a single nucleotide. By assembling these modules, researchers can target any sequence they want.
Studies have shown that this protein can function similarly to zinc finger nuclease, and it is easier to implement and cheaper.
- CRISPR/Cas9 technology
CRISPR/Cas9 is the third generation of "Genome Site Editing Technology" after "Zinc Finger Endonuclease (ZFN)" and "Transcription Activator-like Effector Nuclease (TALEN)". Compared with the previous two generations of technology, its low cost, simple production, fast and efficient advantages, make it quickly became popular in laboratories around the world, becoming an effective tool in scientific research, medical and other fields.
CRISPR/Cas9 is a newly emerged technique of RNA-directed Cas9 nuclease to edit targeted genes. CRISPR/Cas9 is an acquired immune defense mechanism evolved by bacteria and archaea in response to constant attacks by viruses and plasmids. In this system, crRNA combines with tracrRNA through base pairing to form double-stranded RNA. This tracrRNA/crRNA binary complex directs Cas9 protein to cut double-stranded DNA at the target site of the crRNA guide sequence.
In the process of genome editing, tracrRNA and crRNA can be fused into a single RNA (sgRNA) and the expression can also play the role of targeted cleavage.
The advantages of CRISPR/Cas9 are simple operation and high efficiency for the genome. When you need to edit a certain target site, you only need to express the corresponding sgRNA, and you don't need to modify the Cas9 nuclease. It can perform highly efficient targeted editing of the genome of any species. One disadvantage of genome editing technology is the off-target effect, which may cut off the DNA outside the target site, resulting in indel markers.
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