L14 : Cas9, DNA repair, genome editing Flashcards
(52 cards)
What is an example of a signalling pathway triggered by COA production?
CalpL-CalpT-CalpS
In sulfurihydrogenibium
How are Calp genes organised in sulfurihydrogenibium?
Functionally related genes are clustered together in genomic region
- Downstream of Cas10/Csm1
What is the role of COA in CRISPR signalling?
Activates downstream effectors like CalpL
Amplifies the anti-viral response
What is CalpL and what domain does it have?
Lon protease domain
SAVED domain
- Responsible for binding cA4
What does CalpL do upon binding of cA4?
Becomes active in presence of cA4 (not other COAs)
Cleaves CalpT
- Triggering downstream transcriptional effects
What is CalpT and its function?
Regulatory protein
Forms complex with CalpS
Cleaved by CalpL in presence of cA4
What blocks cleavage of CalpT by CalpL in experiments?
A195E mutation in CalpT blocks cleavage by CalpL
What is CalpS predicted to be?
Sigma factor
- Activates transcription
Based on AlphaFold predictions
What is the functional relationship between CalpT and CalpS?
CalpT binds and inhibits CalpS
Prevents interaction with RNAP and DNA promoters
What happens after CalpT is cleaved by CalpL?
CalpS is released from inhibition
- Can activate transcription
- Likely of stress response genes
Where were Calp genes studied and why is direct testing limited?
In bacterium Sulfurihydrogenibium
- cannot be cultured
- cannot be genetically manipulated
- tests rely on expression in model organisms such as E.c
Which of CalpT/S/L and RNAP copurify when expressed in E.c?
CalpS and CalpT
CalpS and E.c RNAP
What does the Type III CRISPR system (Csm) primarily target?
RNA to target and cleave specific RNA molecules
Also has collateral ssDNA cleavage activity
What is the SthCsm complex?
Type III CRISPR complex derived from Streptococcus thermophilus
- Targets RNA degradation or modification
What happens when RNA targeted by SthCsm complex?
Most RNA is degraded but fraction remains
Some of this remaining RNA is unmodified whilst other parts are cleaved and repaired
What type of changes are seen in RNA after SthCsm targeting?
Edited RNAs often show deletions of 6, 12, 18 bps
Consistent with cleavage and repair activity of Csm complex
Why are deletions often 6, 12, 18 bp in size?
Csm complex cleaves RNA at regular 6bp intervals
- Has 3 subunits with cleavage activity
- Evenly spaced cuts can lead to deletions in multiples of 6 bp
Why is it important that deletions are multiples of 3 bp?
Deleting multiples of 3 bp preserves ORF
Allows mRNA to still be translated by ribosome into functional protein
How is cleaved RNA repaired after Csm complex targeting?
RNA fragments are rejoined by cells tRNA splicing machinery
- Specifically RNA ligases
How was it confirmed that tRNA splicinig machinery is responsibe for RNA repair?
Deletion experiments
When tRNA splicing components were knocked down
- RNA repair was reduced
- Confirmed role
What is the therapeutic potential of SthCsm cleavage and RNA repair?
May be used to remove disease-causing mutations from RNA
- Allows partial restoration of gene expression
- Potential treatment of genetic diseases
Does this process resotre mutant RNA to WT?
No
Does not restore original sequence
Removes mutations and rejoins RNA
- Often results in corrected but slightly altered sequence
Give an example of a disease and mutation that could be targeted with Csm deletion approach?
Cystic fibrosis
Eg. W1282X mutation in CFTR gene
- TGG codon becomes TGA (stop)
- Causes nonsense-mediated decay
- Contributes to symptoms
How could Type III CRISPR help with W1282X mutation>
Targeting of premature stop codon with crRNA
Csm complex can cleave and remove stop, RNA ligase rejoins
Potentially restoring partial FL CFTR protein expression
