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.






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).






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