CRISPR Flashcards
what is PAM?
protospacer adjacent motif, necessary for Cas9 cleavage. Way for bacteria to avoid self-targeting
where does Cas9 make cut relative to PAM?
3-4 basepairs upstream
how to choose a good cut site for knockout?
cuts within exon, ideally most 5’ exon that is conserved between splice variants. High on-target, low off-target
how to transfect and select for transformants?
transfect with integrating lentivirus, add puromycin to select for cells that don’t have
how to identify edited cells?
western blot to assay KO, PCR/sequencing, or if edited cells have clear phenotype you can screen for it, or do immunofluorescence
how does CRISPRi work?
dead Cas9 targets to complementary site, then sticks there, acting as a steric hindrance to RNA pol or used to recruit transcriptional repressor proteins (ie KRAB)
how does CRISPRa work?
dead Cas9 targets to complementary site, recruits transcriptional activators (like VP64). This effect can be enhanced by the recruitment of multiple factors (such as aptamers, antibodies, whatever)
how can engineered Cas9 be used to address epigenetics questions?
can link a cytidine deaminase to Cas9, can fuse histone modifier affecting expression, and can label a targeted region with GFP-tagged Cas9
what are the 3 components of crispr array?
Cas genes, spacers, and repeats
how many spacers can a bacteria have?
as few as 1, as many as 450+
what is pre-crRNA
transcribed from leader sequence upstream of spacers/repeats, then matured into multiple, shorter segments, processed by either a Cas or RNAse III depending on system
