Question

Please describe how you can down regulate transcription of specific genes using CRISPR technology

Answer 1

CRISPR is a short form of Clusters of Regularly interspaced Short palindromic Repeats. This is actually a specialized region of DNA in bacteria to protect their genome from invasions by viral DNA. As the name suggests, there are nucleotide repeats that are distributed throughout the CRISPR gene with interspersed nucleotides as spacers. This technology works by the production of RNA form the CRISPR sequence (crRNA) which forms complementary base pairin with the invaded sequence. this complex is then recognized by an enzyme Cas9 which binds the crRNA along with other RNA (tracrRNA-transcating crRNA) which directs cas 9 to make cuts at the desired locations in the genome. after the cuts are made, the DNA repair mechanism of the cell gets activated to repair the damage.

This technology can be effectively used to downregulate the transcription of specific genes. Knowing the DNA sequence of the target gene to be downregulated, we can construct a crRNA complementary to that sequence either entire sequence or a part of it depending whether we want to cut/modify a part of this gene or to knockout the entire gene). Then the crRNA along with the tracrRNA (together called as guide RNA) along with cas9 protein is inserted into the cell (keeping in view that the sequence against which crRNA is made is unique and present only in this gene). the guide RNA binds the target and gets in cas9 which cuts DNA at the specific sites. then the cells'own DNA repair mechanism will act which will introduce the mutations or other changes in the target gene.