CRISPR Cas immune systems Flashcards
What was identified in 1987 on CRISPR?
Upstream of alkaline phosphatase was a CRISPR locus. Discovered by Ishino et al.
What is the leader in CRISPR?
A promoter for transcription
What is the spacer?
DNA sequence inbetween the repeats
What is the repeats?
repeating sequences spaced out by spacers
What is the timeline of CRISPR discovery?
2002-CRISPR named. Cas genes noted. First hypotheses of CRISPR-Cas function in DNA repair. 2005 CRISPR sequences match virus/plasmids. 2007 CRISPR acquired resistance viruses in Streptoccocus. 2014/15 genome editing started.
What is an example of a CRISPR locus
Associated with lots of Cas genes. All on one side of the leader. Upstream names are CasABCDE123. Alkaline phosphatase gene.
What was shown in 2007?
That the CRISPR spacers provide immunity to invader DNA MGEs (MGE-mobile genetic element, e.g., phage, plasmid etc.) S. thermophilus cultures showed resistance to phage, corresponding to presence of CRISPR DNA spacers matching phage DNA sequences. The prokaryotes could use the virus after infection to change their spacers leading to immunity.
What else was shown about the spacers in 2007?
Spacer numbers match various parts of the phage genome that infects it. The Cas5 inactivation resulted in loss of the phage resistance and perhaps Cas5 acts as a nuclease because it contains an HNH-type nuclease motif. (Cas5 now called Cas9)
What do Cas genes A-E form that was discovered in E. coli?
A complex called CASCADE (CRISPR associated complex for antiviral defence.
What did they find in a northern blotting experiment of different cas genes?
Extracted total amount of RNA from WT cells. Delete different Cas. knockout CasA see CRISPR molecules formed same with CasB slightly less with CasC. Nothing formed with CasD. Cas E showed lots of big RNA suggesting CasE is responsible for chopping up the RNA. Did the same but with Cas proteins and got the same results.
What happened when CasE was overexpressed in preCRISPR RNA?
Generated a synthetically produced RNA molecule. Big blob of preCRISPR RNA was converted to smaller lines on western blot.
What are the steps to northern blotting(Read up on for exam)?
Gather sample, electrophoresis, membrane transfer, labeled probe detection, target detection.
How is pre-crRNA transcript converted to crRNA?
cascade-cRNA complex targets complementary DNA to form an R loop. Pre-cRNA transcripts are autonomously folded into hairpin structures these are targeted by the active site of CasE.
How does the ferredoxin fold on CasE/Cas6 ribonucleases allow the biogenesis of CRISPR RNA?
Ferredoxin fold is a conserved structural motif characterized by two antiparallel beta sheets surrounded by alpha helices. Progressively cuts them up until hair pin loop is formed.
