Lecture 4 Flashcards
What is genome editing?
A form of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using nucleases.
What does CRISPR stand for?
Clustered Regulatory Interspaced Short Palindromic Repeats
Describe CRISPR as an adaptive immune regulator.
CRISPR acts as an immune system in prokaryotes against invading DNA/RNA.
Invading DNA is recognised and cut by Cas1-Cas2 protein complexes into fragments termed protospacers.
Protospacers integrated into CRISPR locus located in the bacterial genome.
Upon viral reinfection, transcription of the protospacers to RNA is activated which bind to Cas9.
Cas9/RNA duplex is recruited to complementary sequence on the invading strand of DNA.
Cas9 cuts DNA strands creating a double strand break to prevent infection.
Describe the structure of the CRISPR locus from the 5’ end to the 3’ end.
tracrRNA > operon of cas genes encoding Cat protein components > identical repeat arrays with invading DNA in between each spacer.
What does the guide RNA direct in CRISPR?
Cas9 protein to target a specific sequence.
What is the Cas operon?
Encodes Cas proteins required for DNA cleavage.
Describe Cas9.
Type II Cas protein.
REC lobe = REC1 and REC2 domains recognise DNA/RNA duplex
NUC lobe = HNH domain cleaves DNA complementary to crRNA. RuvC domain cleaves DNA non-complementary to crRNA. PI domain interacts with the PAM sequence.
What dictates whether the Cas9 engaged with the DNA sequences cleaves the DNA?
PAM sequence
If there is no PAM downstream from the complex, it won’t cleave.
Describe engineers CRISPR/Cas9 for biomedical studies.
crRNA and tracrRNA linked by adding a linker loop to create a single component gRNA.
Deposition of the Cas9/gRNA complex at a desired locus of the genome will enable site-specific cleavage through nuclease activity.
The repairs of the DNA break by endogenous DNA repair pathways enables specific genomic edits to be introduced.
What should the gRNA sequence contain for selective CRISPR positioning and DNA cleavage?
Contain a proton-spacer sequence (target sequence) upstream of the PAM site.
gRNAs should be selective to a single genome locus to avoid off target effects.
What is the PAM site for Cas9?
NGG
What is critical for desired CRISPR-mediated DNA editing?
Cellular DNA repair pathways.
When Cas9 clears DNA, a double strand break is introduced.
HDR or NHEJ pathways function downstream to repair DNA, creating the desired genetic edit.
What type of DNA repair is key for CRISPR knock out studies?
Non-homologous end joining (NHEJ).
Does not use a homology template to repair DNA, so error prone.
Fills the break with random base pairs so random INDELs change regions in the reading frame and can introduce premature stop codons in the target sequence of DNA.
What type of DNA repair is key for CRISPR knock in studies?
Homology-directed repair (HDR).
DNA is precisely repaired using sister chromatid during S phase of cell cycle so sequence of the region is completely preserved.
Describe CRISPR mediated knock out via NHEJ.
Target Cas9-gRNA complex to gene of interest.
DSB introduced.
Cell repairs the break via NHEJ (error prone).
INDELs introduced generating a frameshift.
Normal gene product not expressed.
