ompATb is a mycobacterium turberculosis gene that enables these bacteria to surv

Question

ompATb is a mycobacterium turberculosis gene that enables these bacteria to survive acidic environments. M. Tuberculosis is a bacterium which typically infects macrophages (a type of immune system cell). To study the function of the ompAtb gene, you plan to create a cell line with non- functional ompATb gene using CRISPR. The crispr nuclease will cut the ompATb gene based on a specific guide RNA, which is complimentary to the sequence of interest.

When the CRISPR nuclease creates a double stranded break in the gene, the repair machinery can introduce a frameshift mutation in the gene (essentially creating a knockout). Based on this description, where would you want the guide RNA to bind?

Explanation / Answer

I want the guide RNA to bind to the coding region of the ompATb gene sequence. When a frameshift mutation is introduced in the coding region, then the resulting amino acid sequence which is being translated will also change. This leads to the creation of complete non-functional ompATb gene, essentially creating a knockout. It becomes very difficult for the microbes to survive under acidic pH when the ompATb gene becomes non-functional.

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