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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).

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A single nucleotide polymorphism (rs10065172 SNP) was reported for t.docx

  • 1. 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.
  • 2. 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).