L12 : Cas9, DNA repair, genome editing Flashcards
(44 cards)
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
Where is CRISPR found?
Genomes of bacteria and archaea
Organised as arrays of short DNA repeats interspersed with unique spacer sequences
What is required for genome editing?
Must introduce targeted double strand break at desired location in genome
What is the importance of CRISPR/Cas9 in genome editing?
Revolutionary system
Allowed programmable method for efficient introduction of precise mutations within genome
Briefly describe discovery of CRISPR in bacteria
1987
- 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
What is a plaque assay?
Involves infecting law of bacteria with phage on agar plate
If susceptible, phage replicates and form plaques (clear zones)
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. Resistant bacteria will not form plaques
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
What were the conclusion from Strep modification experiment?
Proved spacer sequences directly confer resistance
Established 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 via CRISPR-Cas?
- SPACER ACQUISITION
Insertion of short DNA fragment into CRISPR array - crRNA BIOGENESIS
Transcription and processing into mature crRNA - INTERFERENCE
Targeting of Cas9 RNA guided nuclease (by crRNA) to foreign DNA and specific cleavage of recognised DNA
How are CRISPR systems classified?
CRISPR mechanisms follow common pattern
Different evolutionary lineages discovered and classified into two broad classes
What are the features of Class 1 CRISPR systems?
- Multi subunit effector complexes (containing 4-7 Cas proteins)
- Prevalence: most common, 90% identified CRISPR loci
- Hosts: found in bacteria and archaea
- More complex and harder to adapt for genome editing
What example types does Class 1 and 2 systems include?
Type I
Type III
Type II (Cas9)
Type V (Cpf1/Cas12)
Type VI (Cas13)
What are the features of Class 2 CRISPR systems?
- Single subunit effectors
- Host: almost exclusively in bacteria
- Prevalence: less common
- Preferred in biotech
What are features of the process of spacer acquisition?
- Rare and infrequent
- Occurs preferentially during DNA replication/repair, or near replication forks
- Bias towards non-self targets (self targeting = lethal)
- Can be primed or unprimed (do novo)
- New spacers added next to PAM
How common is Cas1 and Cas2?
Core set of proteins conserved across most systems
Form Cas1-Cas2 complex
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)
Where does spacer integration occur in the CRISPR array?
Leader-proximal (5’) end of array
- New spacers inserted between leader and first repeat
- Preserves order of acquisition
What is the molecular mechanism of spacer integration?
- 3’-OH of protospacer performs two nucleophilic attacks
- One at leader end of repeat
- Second at spacer end of repeat - This causes insertion of spacer between the repeats
- Template for repeat ends up on each side of new spacer
- DNA repair and ligation, completed the insertion
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 and incubation 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
