DNA hypermethylation (an excess of methylation) is associated with many neurological disorders, including a potential role in Alzheimer's disease. a. When comparing individuals with and without Alzheimer's, which of the following 'omics techniques (exome sequencing, whole genome sequencing, transcriptomics, or proteomics) would you expect to be the most informative if your goal is to locate potentially causative methylation differences? Briefly justify your answer. b. The pdCas9-Tet1-CD enzyme (Xu et al Cell Discov. 2016) fuses an enzyme that can carry out cytosine demethylation to a mutant version of the CRISPR/Cas9 enzyme that localizes to DNA using a guide RNA in exactly the same manner as we discussed in class for standard Cas9, except that this version does not cut the genome. This demethylase enzyme can then act to remove DNA methylation proximal to where it is stably bound. Imagine that you have identified a gene that is hypermethylated specifically in patients with Alzheimer's patients. In which region (1-4) of the map below would you target your guide RNA to have the greatest chance of success? Briefly justify your reason. Choose only one region. The exons and introns noted represent the protein coding region of the gene; the forward arrow indicates the transcriptional start site and points in the direction of transcription. Region 1 Region 2 Region 3 Exon1 Intron 1 Exon 2 Intron 2 Exon 3 Region 4 c. Briefly describe one consideration you would take into account when choosing the specific sequence to target with your guide RNA to ensure correct and specific localization of the pdCas9-Tet1-CD complex. d. If you were to apply for permission to begin a clinical trial for a treatment based on your demethylation experiment above, what is one potential safety-related concern that a regulator might bring up?

DNA hypermethylation (an excess of methylation) is associated with many neurological disorders, including a potential role in Alzheimer's disease. a. When comparing individuals with and without Alzheimer's, which of the following 'omics techniques (exome sequencing, whole genome sequencing, transcriptomics, or proteomics) would you expect to be the most informative if your goal is to locate potentially causative methylation differences? Briefly justify your answer. b. The pdCas9-Tet1-CD enzyme (Xu et al Cell Discov. 2016) fuses an enzyme that can carry out cytosine demethylation to a mutant version of the CRISPR/Cas9 enzyme that localizes to DNA using a guide RNA in exactly the same manner as we discussed in class for standard Cas9, except that this version does not cut the genome. This demethylase enzyme can then act to remove DNA methylation proximal to where it is stably bound. Imagine that you have identified a gene that is hypermethylated specifically in patients with Alzheimer's patients. In which region (1-4) of the map below would you target your guide RNA to have the greatest chance of success? Briefly justify your reason. Choose only one region. The exons and introns noted represent the protein coding region of the gene; the forward arrow indicates the transcriptional start site and points in the direction of transcription. Region 1 Region 2 Region 3 Exon1 Intron 1 Exon 2 Intron 2 Exon 3 Region 4 c. Briefly describe one consideration you would take into account when choosing the specific sequence to target with your guide RNA to ensure correct and specific localization of the pdCas9-Tet1-CD complex. d. If you were to apply for permission to begin a clinical trial for a treatment based on your demethylation experiment above, what is one potential safety-related concern that a regulator might bring up?