Question

Control of gene expression is thought to be achieved (in part) by establishing loops of chromatin such that genes are influenced by DNA enhancers that lie within the same “loop” of DNA . Insulator elements define the boundaries between these distinct chromatin domains. Suppose cells have a region of the chromosome that looks like the following:

Gene B B' Gene A A' Enhancer A Enhancer B Insulator

Enhancer A’ helps drive expression of Gene A exclusively in the kidney. Enhancer B’ helps drive expression of gene B exclusively in the liver. A/A’ lie on one side of an insulator element and B/B’ lie on the other side. You hypothesize that gene A would be controlled by enhancer B’ (i.e., gene A is expressed in the liver), if you were to alter the topology of the chromosome so that gene A is in the proximity to enhancer B’ in the nucleus while enhancer A and the insulator element were segregated into a distinct “loop“ (that does not contain gene A, Gene B, or enhancer B’). How would you use CRISPR/Cas9-based technology to test this hypothesis?

Answer 1

Background information : CRISPR-Cas9 is a recent , faster , cheaper and more efficient technique of genome editing. It was modified from a naturally occuring genome editing system in bacteria. It is based on capturing of small DNA pieces ( called snippets) from in the invading viruses to make the segments of DNA called CRISPR arrays. These arrays also acts as memory chips, in the case if viruses attacks again ,the bacteria produce RNA segments from CRISPR arrays to target the virus DNA in order to disable it.

How to use CRISPR technology in gene editing:

• Firstly in a lab we need to create a small piece of RNA with a short " guide" sequence that attaches to target sequence (i.e., in the case Gene B and enhancer A) DNA in the genome.
• Then, RNA also binds to Cas 9 enzyme which cuts the DNA at the target location.
• Once, the cutting is done then , the cell own's genetic material and mechanism repairs the DNA by various Dna repair methods like base exclusion repair method, nucleotide exclusion repair method using enzymes DNA polymerases.
• The final hypothesis / or result is checked by protein expression studies.