LIFESCI 7C Lecture Notes - Lecture 13: Cas9, Homologous Recombination, Non-Homologous End Joining
Document Summary
Most cancer mutations are on noncoding regions of dna. Evolutionary: quantifies selective constraint as a proxy for functionality. Region 1 has more expression because it has gfp expressed more than region 2. How does crispr work in the lab: cas9 protein binds to a single-guide rna (sgrna), cas9/sgrna scans dna for a pam sequence, sgrna hybridizes with target dna, cas9 cuts target dna (double-break) When dna cuts, it falls under the the dna repair pathway by either homologous recombination or non- homologous end joining. How does cas9 know where to unwind dna: scan dna for the pam sequence (5 ngg, unwind dna near pam, sgrna can hybridize w/ dna target, cas9 cuts 3 nucleotides upstream. Look at pam sequence on both strands of dna. Cas9 cuts dna 3 bases before the pam sgrna will bind to the opposite strand in the opposite sequence. Filtration, reabsorption, secretion, loop of henle, renal corpuscle proximal.
