Gene Editing means to edit genes or our DNA, for various purposes including correcting a mutation. Also, inserting or adding gene which will give us desirable characteristics, i.e., improving crop yield and quality.
Moreover, making synthetic microorganism for the production of fuels and for degrading various normally non-degradable able materials. Therefore, to be able to carry out these experiments, three gene-editing tools are developed.
The three genetic tools are;
- Zinc finger nucleases (ZFN),
- Transcriptional activators like effector nucleases (TALENS)
- Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).
What is Zinc finger nucleases (ZFN)?
ZFN was discovered in 1990 and is the first gene-editing tool. Its principle is based on DNA binding, followed by cleaving that target region.
ZFN consist of two components; 1) DNA binding domain. And 2) DNA cleavage domain. These two domains are joined together to make ZFN.
DNA binding domain is also called the Zinc finger domain. It is named because it has DNA binding zinc proteins which are finger-like shaped. There are several zinc finger proteins or repeats. Each repeat recognizes three nucleotide bases. Typically, a ZFN contains about three to nine zinc finger repeats each identifying three bases.
DNA cleavage domain contains nucleases like FOK1 from Flavobacterium. It recognizes the DNA binding domain, and cleaves were it is attached.
How ZFN Works?
ZFN works in dimeric form, each attached at one strand of DNA. Along with the DNA binding domain, the DNA cleavage domain is also assigned.
It usually is not in dimeric form. But upon recognizing the binding site, it dimerizes and activates fok1 nucleases, which then cleaves the DNA.
ZFN is used both as for gene knockouts as well as for insertion of genes. For inserting a gene, we will add the sequence of that gene along with ZFN machinery. And after cleavage, based on homology-directed repair system of the cell, it will incorporate it into the DNA.
ZFN PROS AND CONS
- The pros are as it is the first gene-editing tool, so, it paved the way to new horizons in genetic engineering. Also, enabled us to learn quite a lot about genetics, genetic engineering, genes knockouts, and gene knock-ins.
- The cons are that they have a very high ratio of off-target mutations and cleavages. Because each zinc finger repeats recognizes only three bases making it error-prone. Moreover, ZFN is foreign to the body; studies have been shown in animal models that it causes immunogenicity.
What is TALENS?
TALENS like ZFN is made of two parts 1) transcriptional activator-like effector protein and 2) DNA cleavage domain.
Transcriptional activators like effector (TALE) are proteins which act as transcriptional activators. Their mode of action is the same as them and also bind to a specific sequence in the DNA.
TALE proteins are present in bacteria, Xanthomonas, where they bind to a promotor and switch on or off a gene. TALE when used as a DNA binding protein, they attach to a specific single nucleotide.
DNA cleavage domain, just like ZFN contains fok1 nuclease from Flavobacterium.
How TALEN Works?
First DNA binding domain binds to a target, where each TALE protein attached to a single specific nucleotide. Then it attracts the DNA cleavage domain, both domains dimerize, followed by cleavage.
The target site of TALEN is composed of over 20 base nucleotides, usually 30~40 bases.
TALENs PROS AND CONS
- Its pros are that it is simpler than ZFN and binds to only single nucleotide rather than three bases. Also, it is less complicated than ZFN.
- Its cons are that they are challenging to engineer and it has problems in the delivery system. Moreover, like ZFN it also generates an immune response.
- Also, TALEN is time, effort and cost consuming.
What is CRISPR?
CRISPR is the latest among these three gene-editing tools.
It derived from bacteria, where it provides adaptive immunity to it after a viral invasion. Bactria does so by capturing a piece of its DNA and then integrating into its genome. Later, transcribing it if the same virus attacks again. The transcribe RNA on the basis of complementarity binds with foreign DNA. And singles bacterial CRISPR machinery to cleave it.
CRISPR machinery consist of Cas proteins, sgRNA, Spacer Sequence, PAM sequence and CRISPR locus.
Explore More about CRISPR: What is CRISPR Cas9?
How CRISPR Works?
CRISPR works by first designing spacer sequence, which is complementary to our target sequence. The spacer sequence will then be joined with synthetically guide RNA.
The spacer-sgRNA hybrid will then be introduced into the target sample along with Cas proteins. It can be Cas9 or Cas13 or any other Cas protein depending on the need for experiment.
sgRNA hybrid will recognize the PAM sequences and binds to the target. Once annealed, it signals Cas proteins, followed by cleavage.
Video: CRISPR – Gene editing and Beyond
CRISPR Pros and Cons
- Its pros are that it is fast and cheap as compared to the other gene-editing tools. Moreover, it can design easily.
- Its cons are that it also has a high percentage of off-target mutations.
Explore More: Scientists find new and smaller CRISPR gene editor: CasX
Comparison between ZFN, TALEN, and CRISPR
Also Read: 5 facts and Myths about CRISPR