BIO260 Principles of Genecs Applicaon Acvity 3: DNA Repair This acvity can be co

BIO260 Principles of Genecs Applicaon Acvity 3: DNA Repair This acvity can be completed individually or in small groups of two to three people. PURPOSE The purpose of this acvity is to simulate the process that a scienst in a research lab would use to develop an assay to use CRISPR/Cas9 for gene eding. This would be useful to repair a genec mutaon involved in the development of a genec disorder or cancer. Addionally, you will explore the bioethical implicaons of using CRISPR/Cas9 for genec engineering. Skills TASK You will use the following skills to help you design your crRNA: – Retrieving and analyzing DNA sequences for gene targets – Idenfying PAM sites in a DNA sequence – Communicang your raonal for selecng a specific PAM to design your crRNA Knowledge You will gain the following knowledge: – Familiarity with retrieving DNA sequences from NCBI – Understanding the design of crRNA based, including the idenficaon of PAM sites – Understanding the bioethical issues associated with CRISPR/Cas9 gene eding You are sciensts interested in using CRISPR/Cas9 to edit a mutaon in a gene that is associated with a genec disorder. You will have to retrieve the DNA sequence for your gene, and idenfy a target region. Based on the DNA sequence of your target region, you will Idenfy PAM sites and select one for the design of crRNA. CRITERIA FOR SUCCESS CRISPR/Cas9 can be used to edit DNA sequences, by specifically cung at a target PAM site and providing the cell with a DNA template containing the desired fix. Your crRNA should target you genec region of interest and it should sasfy all of the requirements for crRNA, including its length, proximity to a PAM site, specificity to the desired region, and limited off target effects. Resources Naonal Instutes of Health (NIIH) Naonal Center of Biotechnology Informaon (NIH hps://www.ncbi.nlm.nih.gov of Revised SP23.
260 Principles of Genecs Applicaon Acvity 3: DNA repair Acvity to design crRNA 1. Retrieve the DNA sequence for the target gene from NCBI using this link: hps://www.ncbi.nlm.nih.gov 2. Idenfy a region in your gene to target that is 200 – 250 bp in length. You may choose to idenfy a specific mutaon with that region that you are interested in eding. If so, indicate the site of the mutaon in the DNA sequence. 3. Idenfy all of the PAM sites within the DNA sequence, and highlight the one that you plan to use to design your crRNA. 4. Write out the DNA sequence of your crRNA. 5. Idenfy the CRISPR/Cas9 cut site based on crRNA. Quesons 1. Describe two possible advantages of using HDR over using NHEJ with CRISPR/ Cas9. 2. Is it possible that your crRNA targets genec regions other than the region that it was designed to target? 3. Write three different ideas you have about why CRISPR-Cas9 technology could be useful for gene eding. 4. Would you recommend CRISPR/Cas9-mediated gene eding be used in to fix germ line mutaons or somac mutaons? 5. Should CRISPR/Cas9 be used to edit embryos, even though the genec changes and any errors it produced, will be passed on to their biological children?
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Discipline: Principal to genetics