Question

Briefly describe in point form how a geneticist would figure out how a gene works?

Answer 1

One of the best technique that the geneticists follow to find the function of a novel gene is KNOCK OUT mutation or by MUTAGENESIS:

1. KNOCK OUT: Knock out is the process of turning off a gene by inserting a DN fragment into the coding region of the gene. If a DNA fragment is inserted, then after the transcription of the gene, the gene will produce an altered product not able to perform its function in the cell. So by looking at the changes in the cell that has been knocked out for a particular gene, the particular function of the gene can be easily found out. One of the best technique of knock out is the CRISPR/Cas system.

                   For doing a knockout using this system-

· A guide RNA is designed (that has a region complementary to the gene of interest).

· The gRNA is complexed with the Cas9 enzyme( a nuclease) with the help of plasmid vectors.

· Additional to this a DNA fragment is also supplied (through plasmid vector) that has its flanking ends homologous to the gene of interest. In between the flanking ends, the DNA fragment that is present –is the DNA that will actually get inserted within the coding region of the gene.

· When all these have been supplied to the cell, the cas9 protein will form a complex with the gRNA and produce a Double-stranded cut in the gene of interest.

· As soon as the cut is made, by virtue of the Homologous Recombinational Repair system the Double-stranded break will be repaired by taking help from that additional DNA fragment(which was also supplied into the cell in conjugation) and the DNA fragment will be placed by the repair system into the coding region of the gene, thus disrupting the function of the gene.

2. Mutagenesis- another approach is to do mutation by using the same CRISPR/Cas system. In this case, just the additional Homologous DNA fragment is not supplied. So the Cas 9 complexed with the gRNA will do its job i.e. it will produce a double-stranded break. This break will then be repaired using Non-homologous end-joining method(NHEJ). This NHEJ is itself error-prone i.e. while repairing the break, it inserts random insertions/deletions at that site. Due to these unwanted In/dels that gene gets mutated and the function is lost.