The capacity to attain the site-specific modifications inside the genome of mammal has widespread purposes for applied as well as primary research. The zinc finger nucleases display scope in improvising the gene targeting efficiency by inserting the DNA having double-strand breaks within the targeted genes, which then regulate the cell’s endogenous homologous recombination mechanism.
Gene targeting using CRISPR/Cas9
CRISPR-Cas9 is a recently developed approach that is abbreviation for ‘clustered regularly interspaced short palindromic repeats’ and Cas associated with protein 9. This system has produced a lot of excitement in the scientific society as it is known to be cheaper, faster, accurate, and very efficient as compared to other existing genome editing methods.
Gene targeting is one of the most critical advantages of the CRISPR/Cas9 system. It aids in not only in targeting the specific genes, but it also aids in maintaining accuracy with less difficulty and consistency throughout the trials. There are two primary elements that lead to this superiority and make this system more straightforward and easier to use. These are reproducible genome cleavage and programmable sgRNA (single guide RNA) cloning.
Gene targeting in embryonic stem cells:
The process in the production of the gene-targeting mice include:
The first step is development of a gene targeting vector from the isogenic genomic DNA
The technique of electroporation for the targeting vector into the ES cells followed by the isolation of the drug-resistant embryonic stem cell clonedetermination of the gene-targeted ES cell clones
Generation of ES cell mouse chimeras forms second last step
The last step is the germline transmission of the gene-targeted allele.
Targeting vectors:
Targeting vectors are linearized prior to the transfection process. It should contain the following:
The vector should have a modified sequence which is homologous to the target gene.
A selection marker should be present, and this aids in selecting the transfected cells and elevates the targeted recombination products. Marker can be a positive or a negative selection marker.
Targeting vector types can be an insertion vector and most widely used replacement vector.
Replacement vector:
It has a homologous sequence, positive and negative selection marker; the double homologous recombination should take place, and flanking vector elements should be excised. The linearization site is observed to be outside the homologous region.
Insertion vector:
The linearization site is known to be within the homologous sequence. The insertion is made into the target site through single reciprocal recombination. It has a 5-20-fold higher frequency as compared to the replacement vector.
Selectable marker:
Selectable marker refers to the gene which is introduced into the cell, particularly a bacteria or to the cells found in the culture, which confers a character suitable for the artificial selection. Selectable markers are mainly antibiotic resistance genes.
Positive selection marker:
It isolates rare but stably transfected cells. It is embedded within the homologous gene in the vector to making the gene non-functional and can be used as a mutagen. For instance, antibiotic resistance permits the host body to survive antibiotic selection.
Negative selection marker:
It aids in the elimination of random insertions as well as the insertion of heterologous elements. For example, thymidine kinase makes the host body sensitive to ganciclovir selection.
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