A single nucleotide polymorphism (rs10065172 SNP) was reported for the human IRGM gene: a “C” to “T” variation at position 313 of IRGM coding sequence (Transcript ID: IRGM-001 ENST00000522154). This exonic synonymous SNP (defined as “313C>T”) has been associated with increased susceptibilites to an inflammatory disease known as the “Crohn’s disease”.

Additional studies suggested that the microRNA-196 may down-regulate IRGM expression from the protective allele (or the “C” allele), but not from the risk-associated allele (or the “T” allele). Loss of the regulation of IRGM expression has been shown to disrupt cellular autophagy.

The hypothesis is that the 313C>T SNP causes a loss of regulation of IRGM expression by microRNA-196.

MATERIALS AVAILABLE: Crohn’s patient cells (genotype: IRGM ^313C/313T), healthy cells (genotype: IRGM ^313C/313C). All reagents for DNA, RNA, protein, and cell work are available.

QUESTION:

Please develop an overall strategy, using concepts learned from “genome editing” lectures, to achieve the goal of genetic engineering of IRGM allele in provided cell lines. A flow chart of experimental procedures, accompanied by detailed description of each step, is necessary. Your approach should be sufficient for you to test the hypothesis. NOTE: please cover topics of Crispr/Cas9, HDR, PAM site, Donor sequence, Detection of Modified Allele, Off-targets, etc. Specifics about microRNA-196 are not essential. Limit your answer to 3 pages (single-spaced).