L12 : Cas9, DNA repair, genome editing Flashcards
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
Briefly describe discovery of CRISPR in bacteria
Observed short repetitive DNA sequence in arrays within genome
- Often next to genes linked to repair
- Sequencing of spacers between repeats show often derived from bacteriophage
- Possible involvement in protection from bacteriophages
- First indication of bacterial immune system
How was Streptococcus resistance to phage investigated?
Plaque assay:
1. Culture of Streptococcus thermophilus
2. Lawn of bacteria on agar plate
3. Addition of phage
4. If phage able to replicate, will observe plaques on plate
5. Surviving bacteria will be investigated
Note: Barrangou 2007
Provide a basic explanation of CRISPR/Cas
- Bacterium infected by bacteriophage
- Cas genes proteins chop up fragments of invading phage DNA
- Makes new repeat and spacer (fragment of phage DNA ~20 bp)
- Incorporated into array of repeats
- Bacteria can use this DNA to recognise phage in future
How was Streptococcus resistance to phage detected and developed?
Detected via plaque assay
- 1 = sensitive
- <1 = resistant
Resistance levels vary between bacteria and phage
Genomic analysis shows resistance coincides with acquisition of new CRISPR spacer sequences from single infection
What were the 4 key experiments in verifying spacer acquisition confers resistance?
Researchers modified CRISPR system in Streptococcus thermophilus
- No modification
- Resistant - Single repeat, no spacers
- Sensitive - Repeat present but disrupted (cassette inserted)
- Sensitive - Reintroduction of spacers S1 and S2
- Resistant again
Showed spacers necessary for resistance
First major evidence for bacterial adaptive immune response
How does the acquired spacer help the bacterium?
Type II CRISPR/Cas system
- Transcription of CRISPR non-coding RNA, forming pre-crRNA
- Processing into indivudal crRNAs (each with one repeat + one spacer)
- Cas9 (RNA-guided nuclease) uses crRNA to recognise matching phage DNA
- Cas9 cleaves phage DNA, neutralising threat
What are the three phases of bacterial adaptive immunity?
- Insertion of short DNA fragment into CRISPR array (new spacer)
- Transcription and maturation of crRNA
- Targeting of Cas9 RNA guided nuclease (by crRNA) to foreign DNA and specific cleavage of recognised DNA
What are the 2 classes of CRISPR systems?
Class 1:
- Multi subunit effector complexes (containing 4-7 Cas proteins)
- Most common, 90% identified CRISPR loci
- Found in bacteria and archaea
Class 2:
- Single subunit effectors
- Almost exclusively in bacteria
What are features of the process of spacer acquisition?
- Rare and infrequent
- Bias towards non-self targets (self targeting = lethal)
- Can be primed or unprimed (do novo)
- New spacers added next to PAM
- Related to DNA ends and DNA repair/replication, restriction systems
How do Cas1-Cas2 proteins integrate new spacers into the array?
Cas1-Cas2 = spacer integrase complex
- Catalyses insert of new spacer at 5’ end of CRISPR array, after leader sequence
- Spacer integration generates new repeat (ready for next spacer)
What is the molecular mechanism of spacer integration?
- Nucleophilic attack by 3’OH of protospacer at leader end of repeat
- Nucleophilic attack by 3’OH of protospacer at spacer end of repeat
- Half of repeat ends up on each side of new spacer
- DNA repair and ligation
How was Cas1-Cas2 integration demonstrated in vitro?
- Express and purify Cas1 and Cas2
- Test ability to insert spacer sequence into plasmid containing CRISPR array
- Addition of combinations of protospacer, Cas1, Cas2, pCRISPR (plasmid)
- Relaxed plasmids observed in the gel when all components present
How was the incorporation of radiolabelled protospacers by Cas1-Cas2 demonstrated
- Express and purify Cas1 and Cas2
- Addition of radiolabelled spacer
- Can follow insertion of protospacer into plasmid
What features of protospacers are important for Cas1-Cas2 spacer integration? Experiment?
- dsDNA protospacers
- 3’OH at both ends of protospacer crucial for integration
- Experimental mutations at 3’ ends reduced or prevented integration
- Confirms nucleophilic attack y 3’OH drives insertion
What did sequencing reveal about in vitro spacer Cas1-Cas2 integration sites
- ~71% occured in CRISPR repeat regions
- Not all insertions at 5’ end of array
- Sometimes occurred in non-CRISPR sites such as cruciform structure in amp gene (other end of plasmid)
How does CRISPR system function in S. pyogenes? With tracRNA?
- CRISPR array transcribed into pre-crRNA
- tracRNA binds to repeat sequences in pre-crRNA
- Processing by RNAse III + Cas9 + tracRNA
- Forms mature crRNA, which guides Cas9 to target phage DNA
What is the role of tracRNA in the CRISPR system of S. pyogenes?
- tracRNA base pairs with repeats in pre-crRNA
- RNAse III + Cas9 cleave both pre-crRNA and tracRNA
- Forms tracRNA:crRNA duplex
- Duplex is loaded into Cas9, guiding to target DNA
crRNA provides targeting sequence but tracRNA essential for proper processing and function
What is PAM and the self-protection mechanism?
PAM (protospacer adjacent motif) is short DNA sequence required for recognising target DNA
Ensures CRISPR/Cas9 does not cleave sequences within CRISPR array
S. pyogenes = NGG
Other Cas9 variants recognise different PAM sequences
What are the three requirements for recognising and cleaving target DNA?
- Complex of crRNA:tracRNA
- 20 bp complementarity between crRNA and target DNA (upstream of PAM)
- Presence of PAM sequence next to target (S. pyogenes = NGG)
Why is the PAM sequence in target DNA?
Not within CRISPR array
Distinguishes self from non-self
What are the nuclease domains of Cas9 and their functions?
RuvC domain
- Cleaves non-target strand
- 3 subdomains dispersed in sequence
- Only 1 active site
HNH-like domain
- Cleaves target strand (complementary to crRNA)
Each cleaves one strand 3-4 bp upstream of PAM sequence, resulting in dsDNA break
What are 2 ways bacteriophages can acquire resistance and reaction from bacteria?
Mutate target site:
Cas9 can tolerate some mismatches in target sequence (particularly distal to PAM)
Mutate PAM:
Mutation of PAM less tolerated
How tolerable is spCas9 in DNA targeting?
S. pyogenes Cas9 (spCas9)
Can tolerate mismatches between crRNA and target DNA
- Up to 5 mismatches possible
- Dependent on position: distal from PAM allows more tolerance
- Recognises and cleaves both NGG and NAG PAMs
