L13 : Cas9, DNA repair, genome editing Flashcards
How does Cas9 use crRNA to cleave target DNA? (recap)
- crRNA guides Cas9 to complementary target DNA
- PAM sequence (eg. NGG) found on non-target strand
- Cas opens DNA, forming DNA:RNA hybrid with target strand
- DNA strands split into separate channels
- HNH cuts target strand
- RuvC cuts non-target strand - Arginine residues (R1333 and R1335) interact with major groove at PAM to help stabilise and position DNA
What is Cas13 and how does it cleave RNA (cis and trans)?
Cas13 is CRISPR system enzyme for targeting and cleaving RNA
- HPEN RNAse domain
- Guided by crRNA (no tracRNA required)
Cis: cuts specific target RNA
Trans: once activated, collaterally cleaves other nucleic acids nearby
What are 2 potential applications of the Cas13 system?
Enables rapid transcript depletion
- Useful for knockdown experiments
Collateral cleavage
- Diagnostic tools
How can CRISPR Cas13 be used in diagnostic tests?
Detects specific RNA transcripts related to pathogen (eg. SHERLOCK)
- Design CRISPR RNA that will base pair with transcript
- Addition of short RNA reporter with fluorescent group and quencher group at either end
- Intact RNA has fluorophore and quencher in closer proximity, quenching fluorescence
- When collateral RNAse activity activated, cleavage between fluorophore and quencher
- Produces fluorescent signal
Technique probably emerge more in coming years
Do all CRISPR systems use the same Cas enzymes?
- Some CRISPR systems share core Cas enzymes
- Others use unique single effector proteins (eg. Cas13, Cas9)
What are the 3 main features of Type III CRISPR system?
- Multiple effector protein subunits in core
- Mostly recognise RNA
- Can cleave RNA and DNA
What are the 4 classes of Type III systems?
Type III A:
- Csm complex (Csm1-5)
Type III B:
- Cmr complex (Cmr1-6)
Type III C:
- Cmr complex (Cmr1-6)
Type III D:
- Csm complex (Csm1-5)
What are the 5 key features of Csm/Cmr complexes?
- RNA guided nuclease complex
- crRNA has 8nt tag at 5’ ed encoded by CRISPR repeat
- No tracRNA, single RNA guiding nuclease complex - 30-45 nucleotide guide sequence
- RNA cleaved by Csm3/Cmr4
- Non specific ssDNA cleavage by Csm1/Cmr2 HD domain
- Cyclic oligoadenylate production by Csm1/Cmr2 PALM domain
- Second messenger, activates diverse downstream enzymes
Describe the structure and roles of the Csm complex?
S. thermophilus
- Csm 1, 2(2), 3(3), 4, 5
- Csm 3 (3 subunits within complex) performs RNA cleavage
- Potential for RNA to be cut at 3 different sites
- Csm1 for ssDNA cleavage and cyclic oligoadenylate production
How is spacer acquisition done in Type III CRISPR systems?
Two potential spacer sources:
1. DNA
2. RNA (if reverse transcriptase activity present)
Inserting spacers into CRISPR array mediated by Cas1-Cas2
Briefly describe the process of Csm assembly and CRISPR transcription?
Assembly of Csm subunits into larger effector complex
- Recognise and cleave RNA (and DNA)
Transcription of CRISPR RNA into long RNA
- Chopped into indivudal crRNAs by Cas6
Why indiscriminately remove ssDNA?
- Csm complex. uses RNA to recognise transcripts (+ during RNA pol transcription)
- Limited DNA region surrounding RNA pol is unwound, revealing ssDNA
- DNA nearby target RNA is likely derived from bacteriophage
How can CRISPR-Csm complex be used for RNA localisation in cells?
Example system: Streptococcus thermophilus Csm
Potential application: Localisation and visualisation
Method:
1. Express all Csm components
- Separate promoters or ORFs separated by T2A peptide (RNA restarts translation at each gene)
2. Mutate active site to disable cleavage activity
3. Label complex with GFP for fluorescence
4. Complex binds specific RNA targets, allowing visualisation of RNA localisation in live cells
How was Csm complex used to visualise Xist RNA in live cells? Limitation?
Xist is abundant ncRNA involved in X chr inactivation
1. Csm complex programmed with crRNA to bind Xist RNA
2. Tagged with GFP to allow live cell imaging
3. Successfully detect Xist foci in cell nucleus
Less effective for diffuse or low-abundance transcripts
- Csm complex fluorescent on its own so signals can overlap and interfere
What are COAs and how are they involved in CRISPR signalling?
Cyclic oligoadenylates
- Produced by Cas10/Csm1 (PALM domain) in Type III systems
- Made by cyclising ATP molecules (eg. cA4 = 4ATPs in cyclic arrangement)
- Act as second messengers to activate diverse enzymes (RNAse, DNAse, transcription regulators
What experiment showed that Type III Csm complex produces COAs upon RNA recognition?
- Csm complex + 205 bp RNA (radioactively labelled) + crRNA + ATP (radioactive)
- Target RNA will produce various fragments depending on the 3 cleavage sites
- Over time, generation of cyclic oligoadenylate will occur
- Coincides with cleavage of target RNA
Conclusion:
- Recognition of target RNA leads to production of COA
- Only want additional bacteriophage response following recognition of foreign RNA
How do COAs activate signalling cascade in Sulfurihydrogenibium
- cA4 (cyclic tetra adenylate) produced upon target RNA recognition
- Binds to protein complex of 3 components
CalpL (protease)
- Activated CalpL cleaves CalpT, releasing CalpS
CalpT (repressor)
- Binds CalpS to inhibit activity
CalpS (sigma factor)
- Binds RNA pol to intitiate transcription
- Likely triggers stress-related transcriptional response
- Not fully understood
