CMMB 403 Final Exam with complete solution

CMMB 403 Final Exam with complete solution what does CRISPR use in order to generate changes in genome? is CRISPR forward or reverse genetics? - answer- nuclease - 20 nucleotide RNA sequence to target specific sites in the genome - reverse genetics what is the role of Cas9 in CRISPR - answerendonuclease that catalyzes double stranded breaks what are the two repair paths cells can use to repair double stranded breaks from CRISPR-Cas9 - answer1. non-homologous end joining (NHEJ) - naturally try to repair - imperfect, creates indels causing frameshift mutation 2. directed homology repair - sequence homology on 5' and 3' ends to encourage homologous recombination flanking the double stranded breaks - insert known sequences to try and treat genetic diseases explain the method of conditional gene inactivation - answer- make targetting construct in lab - electroporate construct into embryonic stem cells to target endogenous Shox2 locus - mice generated by blastocyst injection into ESCs carry loxP sites therefore can delete Shox2 2 components for a conditional knockout mouse - answer1. transgenic mice express Cre recombinase in the tissue of interest 2. mice with loxP sites on each side of the gene or region to be deleted How is CRISPR-Cas9 being used in a patient in the USA to treat sickle cell anemia? - answerby inactivating an enhancer of the BCL11A gene (BCL11A gene represses fetal hemoglobin) how does the BCL11A protein affect global gene expression - answerit represses fetal hemoglobin gene expression In what kind of animals is CRISPR good? - answerany animals for which one cell embryos are accessible - good for mammals, not chickens what is the result of CRISPR inactivating Rspo2 in half a frog? - answerRspo knockout= limbless side inactivated by CRISPR had no limb what are transgenic mice good for? - how are they made? - is this targeted? - what are they not useful for? - answergood for mis-expressing genes, enhancer reporters, etc. - generated by pronuclear injection; DNA construct is incorporated randomly into the genome - not useful for LOF because not targeted how are knockout mice made? - is it targeted? - what kind of species is this successful in? - answercreated by genomic targeting in embryonic stem ce