FN - CRISPR-Cas I & II Flashcards
(19 cards)
What is the function of the CRISPR-Cas system in bacteria? (3)
- It is the only known adaptive immune system in bacteria
- Acquires and retains memory of viral infections
- Defends against foreign DNA/RNA by cleaving it
What are the 3 key stages of the CRISPR-Cas mechanism? (3)
- Adaptation – Acquisition of foreign DNA into the CRISPR array as spacers
- Expression & Processing – CRISPR array is transcribed and processed into mature crRNA
- Interference – crRNA guides Cas protein to target matching DNA/RNA for cleavage
What is a PAM (Protospacer Adjacent Motif) and why is it important? (4)
- Short sequence next to the target DNA
- Allows CRISPR to distinguish between self and non-self
- Required for Cas nucleases (e.g. Cas9’s PAM = NGG) to cut
- Not present in bacterial CRISPR array
What role do Cas1 and Cas2 play in adaptation? (3)
- Recognize invading phage DNA
- Excise protospacers and insert them into the CRISPR array
- Store memory for future targeting
How does Cas9 mediate interference in type II systems?
- Uses a complex of crRNA + tracrRNA (or sgRNA)
- Targets matching DNA adjacent to a PAM
- HNH and RuvC nuclease domains generate a blunt double-strand break
What are Class 1 and Class 2 CRISPR systems distinguished by? (2)
- Class 1 – Multiprotein effector complexes (e.g. Type I, III, IV)
- Class 2 – Single protein effectors (e.g. Type II - Cas9, Type V - Cas12, Type VI - Cas13)
What defines Type I CRISPR systems (Class 1)? (3)
- Use Cascade complex + Cas3
- RNA-guided DNA targeting
- Cas3 unwinds and degrades DNA
What defines Type III CRISPR systems (Class 1)? (4)
- Use Cas10-Cascade complex
- RNA-guided RNA and DNA targeting
- Uses cyclic oligoadenylates (cOA) for abortive infection
- Strong system in Mycobacterium tuberculosis
What defines Type IV CRISPR systems (Class 1)? (3)
- Use Csf-Cascade
- Less well characterized
- Also originate from casperon
What defines Type II CRISPR systems (Class 2)? (4)
- Use Cas9 from Streptococcus pyogenes
- RNA-guided DNA targeting
- Requires PAM for activity
- Two domains: RuvC and HNH
What defines Type V CRISPR systems (Class 2)? (4)
- Use Cas12
- RNA-guided DNA targeting
- Cuts with staggered dsDNA breaks
- Can trigger collateral cleavage of single-stranded DNA
What defines Type VI CRISPR systems (Class 2)? (4)
- Use Cas13
- RNA-guided RNA targeting
- Triggers collateral RNA degradation – including host RNA
- Leads to cell death (abortive infection)
What is CRISPR collateral damage and why is it important? (5)
- Non-specific nucleic acid cleavage triggered after target recognition
- Acts as a last-resort defense – kills infected cells to protect population
Types:
- Cas13 (Type VI) – RNA → RNA collateral damage
- Cas12 (Type V) – DNA → DNA collateral damage
- Cas10 (Type III) – RNA → RNA and DNA collateral damage
What is a key mechanism behind CRISPR collateral activity in Type III systems? (3)
- Cas10 synthesizes cyclic oligoadenylates (cOA)
- Activates Csm6 to cleave host RNA
- Can cause dormancy or cell suicide depending on context
How does precision genome editing use CRISPR? (3)
- Cas9/Cas12 are used to generate double-stranded breaks
Two repair pathways:
1. NHEJ (Non-homologous end joining) – error-prone
2. HDR (Homology-directed repair) – high precision with donor templates
What are base editors and how are they used in CRISPR? (3)
- Fusion of deaminases with Cas proteins
- Enable precise single-nucleotide edits without double-strand breaks
- Used in HDR pathway
What are CRISPRi and CRISPRa?
- CRISPRi – dCas9 fused to repressors blocks transcription
- CRISPRa – dCas9 fused to activation domains turns genes on
How has CRISPR been used in diagnostics? (3)
- Cas13 (SHERLOCK) – detects RNA via collateral cleavage
- Cas12 (DETECTR) – detects DNA via collateral cleavage
- Activated nucleases cleave reporter molecules → signal detection
What are Anti-CRISPR proteins (Acrs) and what do they do? (3)
- Produced by phages to evade CRISPR
- Inhibit Cas DNA binding or cleavage
- Can be used to reduce off-target effects and control CRISPR systems
