L13 : Cas9, DNA repair, genome editing

Question 1

What is Cas13 and key features?

Answer 1

Cas13 is CRISPR system enzyme for targeting and cleaving RNA
- HPEN RNAse domain
- Guided by crRNA (no tracRNA required)

Question 2

Describe cis and trans RNA cleavage by Cas13?

Answer 2

Cis: direct cleavage of specific target RNA

Trans: once activated, collaterally cleaves other nearby nucleic acids in non-specific manner

Question 3

What are 2 potential applications of the Cas13 system?

Answer 3

Enables rapid transcript depletion
- Useful for knockdown experiments

Collateral cleavage
- Diagnostic tools

Question 4

What is the general idea of Cas13 in diagnostics?

Answer 4

Cas13 trans-cleavage activity can be used to detect the presence of specific RNA (eg. from virus or pathogen)

Technique probably emerge more in coming years

Question 5

What is an example of how Cas13 could be used in diagnostic tests?

Answer 5

SARS-CoV-2 RNA
Point-of-care diagnostics (eg. SHERLOCK)

Question 6

How can CRISPR Cas13 be used in diagnostic tests?

Answer 6

1. Design CRISPR RNA that will base pair with transcript
2. Addition of short RNA reporter with fluorescent group and quencher group at either end
3. Intact RNA has fluorophore and quencher in closer proximity, quenching fluorescence
4. When collateral RNAse activity activated, cleavage between fluorophore and quencher
5. Produces fluorescent signal

Question 7

Advantages of Cas13 in diagnostics?

Answer 7

• Rapid
• Highly sensitive
• Doesn’t require amplification

Question 8

How are all CRISPR systems similar?

Answer 8

Share same common logic
- Acquisition of foreign sequencers, processing into crRNA, and targeting

Question 9

Do all CRISPR systems use the same Cas enzymes?

Answer 9

• Some CRISPR systems share core Cas enzymes (Cas1-Cas2)
• Others use unique single effector proteins (eg. Cas13, Cas9)

Question 10

What are the 3 main features of Type III CRISPR system?

Answer 10

1. Multiple effector protein subunits in core
2. Mostly recognise RNA
3. Can cleave RNA and DNA

Question 11

What are the 4 classes of Type III systems?

Answer 11

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)

Question 12

What are the 5 key features of Csm/Cmr complexes?

Answer 12

1. RNA guided nuclease complex
   - crRNA has 8nt tag at 5’ ed encoded by CRISPR repeat
   - No tracRNA, single RNA guiding nuclease complex
2. 30-45 nucleotide guide sequence
3. RNA cleaved by Csm3/Cmr4
4. Non specific ssDNA cleavage by Csm1/Cmr2 HD domain
5. Cyclic oligoadenylate production by Csm1/Cmr2 PALM domain
   - Second messenger, activates diverse downstream enzymes

Question 13

What is the composition of the Csm complex in streptococcus thermophilus?

Answer 13

Csm 1, 2(2), 3(3), 4, 5
As seen in S.t crystal structure
~8 subunits total assembled

Question 14

What is the role of Csm3?

Answer 14

• Csm 3 (3 subunits within complex) performs RNA cleavage
• Potential for RNA to be cut at 3 different sites

Question 15

What domains are responsible for Csm1 function?

Answer 15

HD domain
- ssDNA cleavage

PALM domain
- Cyclic oligoadenylate production

Question 16

Why produce cyclic oligoadenylate in Type III systems?

Answer 16

Acts as second messanger
Activates diverse enzymes downstream
- Modulate other immune responses

Question 17

What is the RNA guide format in Type III-A Csm complex?

Answer 17

crRNA
- 8 nt tag from repeat
- ~27 nt spacer

No tracRNA needed

Question 18

How is spacer acquisition done in Type III CRISPR systems?

Answer 18

Two potential spacer sources:
1. DNA
2. RNA (if reverse transcriptase activity present)

Inserting spacers into CRISPR array at leader-proximal end mediated by Cas1-Cas2

Question 19

Briefly describe the process of Csm assembly and CRISPR transcription?

Answer 19

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

Question 20

How is ssDNA cleaved by Csm complex?

Answer 20

1. Csm complex uses RNA to recognise transcripts
   - Cleaved by Csm3
2. Limited DNA region surrounding RNA pol is unwound during transcription, revealing ssDNA
3. Proximity of ssDNA nearby target RNA allows degradation by Csm1
   - May be likely that DNA is derived from bacteriophage

Question 21

What are the 3 overall functions of the Type III A CRISPR system?

Answer 21

• Cleavage of phage RNA transcripts
• Cleavage of possible phage-derived DNA
• Activating various proteins that will interfere with bacteriophage function

Question 22

What is an example III-A system and its potential application?

Answer 22

Example system:
Streptococcus thermophilus Csm
Potential application:
RNA knockdown/localisation and visualisation

Question 23

How can CRISPR-Csm complex be used for visualising specific RNA localisation in live mammalian cells?

Answer 23

Method:
1. Express all Csm components
2. Mutate active site to disable cleavage activity
3. Label complex with GFP for fluorescence
4. Complex binds specific RNA targets, allowing visualisation

Question 24

How was Csm complex used to visualise Xist RNA in live cells?

Answer 24

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

Question 25

What are limitations of using CRISPR-Csm complex for RNA localisation visualisation?

Answer 25

- Less effective for diffuse or low-abundance transcripts - Csm complex fluorescent on its own so signals can overlap and interfere

Question 26

Which protein subunit is responsible for production of COA in Type III systems?

Answer 26

Cas10/Csm1 Through PALM domain - Catalyses formation of cyclic oligoadenylates

Question 27

What and how are COAs produced?

Answer 27

Made by cyclising ATP molecules eg. cA4 = 4ATPs in cyclic arrangement

Question 28

How are COAs involved in CRISPR signalling?

Answer 28

Act as second messengers to activate diverse enzymes involved in antiviral response eg. - RNAse, DNAse - transcription regulators

Question 29

What experiment showed that Type III Csm complex produces COAs upon RNA recognition?

Answer 29

1. Csm complex + 205 bp target RNA (radioactively labelled) + crRNA + ATP (radioactive) 2. Target RNA will produce various fragments depending on the 3 cleavage sites 3. Over time, generation of cyclic oligoadenylate will occur 4. Coincides with cleavage of target RNA

Question 30

What was concluded from experiment on COA production?

Answer 30

Conclusion: - Recognition of target RNA leads to production of COA - Only want additional bacteriophage response following recognition of foreign RNA

Question 31

What is an example of a signalling cascade activated by COA in a specific organism?

Answer 31

Sulfurihydrogenibium 1. cA4 (cyclic tetra adenylate) produced upon target RNA recognition 2. Binds to protein complex of 3 components (CalpL/S/T)

Question 32

What are the roles of CalpL, CalpS, CalpT?

Answer 32

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