Adaptive and heritable immunity Flashcards
1
Q
What is the bacterial immune system?
A
Restriction endonucleases: The “innate immune” system of bacteria
- Bacteria have learned to “restrict” the possibility of attack from foreign DNA (e.g. Viruses) by means of restriction enzymes
- Digest “foreign” DNA that invades the cell:
- cut pahge DNA into smaller, non-infectious fragments
- 4 types of restriction enzymes (RE) exist:
- Type I: Single multifunctional enzyme
- Type II: Separate nuclease and methylase
- Type III: Separate enzymes sharing a common subunit
- Type IV: cut methylated DNA
- In genetig engineering type II RE are mostly used, cleave dsDNA at recognition site (4, 6 or 8 bp in length) without requiring ATP
- Host DNA is protected by methylation of the respective enzyme recognition site
- >3000 restriction enzymes have been studied, >600 are available commercially and are routinely used for DNA manipulation
2
Q
Why do restriction enzymes not cut the DNA of the bacterial cell that makes it?
A
- Host DNA is protected by methylation of the respective enzyme recognition site
- Methyl groups at the restriction sites:
- Block the restriction enzyme
- Protect the bacterial DNA from being cleaved
3
Q
What does CRISPR do?
A
CRISPR = clustered regularly interspaced short palindrome repeats
- ~ 40% of bacteria and almost all archaea have CRISPR loci
- initially thought to function in gene regulation, replicon partitioning, DNA repair, etc..
- later shown that CRISPR is an adaptive and heritable immune system
- crRNA: CRISPR RNA
- The absence of PAM within the CRISPR array prevents autoimmunity
- CRISPR locus:
- Repeat sequences averaging 32 bp are interleaved by variable spacers of approximately the same size
- Number of repeat-spacer units varies greatly:
- from a few to several hundreds e.g.: thermophilic bacterium Chloroflexus sp. Y-400-fl has 374 spacers units per CRISPR loci
- Range of spacer length 21 – 72 nt
- Conserved leader sequence of several hundred bp is located on one side of the cluster
- CRISPR-associated (cas) genes surround locus
- CRISPR locus:
4
Q
How is Spacer acquisition done in CRISPR?
A
- Distal end of cluster contains “older” spacer
- “newer” spacers accumulate next to leader sequence (strain-specific)
- rapid evolvable system
- host chromosomal DNA content is ~25–50 times greater than the plasmid/phage DNA content. Yet chromosome-derived spacers represented only ~2%–22% of the total spacers acquired
- 100- to 1,000-fold preference in spacer acquisition from plasmid/phage over the chromosome
- (largely unknown) mechanism of self- versus non-self-discrimination during spacer acquisition
- Cas1–Cas2 protein complex (in E. coli) triggers acquisition of new 33 bp spacers at the A/T-rich leader end of the CRISPR locus
- all interactions between Cas1–Cas2 and protospacer DNA involve the phosphate backbone rather than base-specific contacts
5
Q
How is crRNA processed?
A
crRNA: CRISPR RNA
- crRNA is the RNA that allows the recognition of the foreign DNA
- first transcribed into pre-crRNA from the DNA of the bacteria, and then processed into mature crRNA that will be integrated into the Cas complex
- endonucleolytic processing into mature products and ladder-like intermediates
6
Q
What is PAM?
A
PAM: proto-spacer adjacent motif.
- short DNA sequence (usually 2-6 base pairs in length) that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR-Cas9.
- required for a Cas nuclease to cut and is generally found 3-4 nucleotides downstream from the cut site.
- The absence of PAM within the CRISPR array prevents autoimmunity
7
Q
How does crRNA-mediated immunity work?
(effector stage)
A
8
Q
What is the role of PAM in target recognition?
A
- Cas9 searches for PAM and reads GG via two Arg
- +1 phosphate of target strand gets pulled out and interacts with «Phosphate lock» of Cas9
- promotes local duplex melting
- crRNA probes the identity upstream of PAM
- if crRNA/target DNA duplex formed → cleavage
9
Q
How are CRISPR systems classified?
A
Class 1:
- Type I, III and IV
- systems possess multisubunit crRNA–effector complexes
Class 2:
- Type II, V and VI
- all functions of the effector complex are carried out by a single protein, such as Cas9
- Type II occurs only in bacteria, not in Archaea
General:
- CRISPR–Cas systems are much more prevalent in archaea (87% of genomes) than in bacteria (50% of genomes).
- Like other defense systems, CRISPR–Cas loci evolve under strong selection pressure exerted
by changing pathogens
→ results in rapid evolution that is largely uncoupled from the evolution of the rest of the respective genomes.
10
Q
What are possible problems that the CRISPR-Cas system poses?
A
- pose a major barrier to HGT (Horizontal gene transfer), a major driving force of bacterial evolution because many HGT events are maladaptive
- However: HGT occurs frequently and is ongoing
- invading DNA (such as phages) encode mechanisms that inhibit CRISPR-Cas
11
Q
What are CRISPR functions beyond defense?
A
- 1 in 250 spacers is self-targeting
- Form of auto-immunity?
- or: allows control of gene expression
- Type II CRISPR abundant in pathogenic bacteria
- deletion of Cas9 gene reduces virulence
- Cas genes and tracRNA are diff. expressed upon changing environment
- many of the alternative activities are linked to processes occurring at the bacterial envelope
- CRISPR-Cas systems implicated in population behaviors that involve extensive envelope alterations (biofilm formation, fruiting body development)
- e.g.: a spacer within the P. aeruginosa CRISPR array has sequence similarity to a gene within a chromosomally integrated prophage → repress swarming motility and biofilm formation
- given the importance of biofilm formation for antibiotic resistance and pathogenesis in P. aeruginosa, it is likely that this CRISPR-Cas system plays an important role in mediating infection of eukaryotic hosts
→ Does CRISPR-Cas mediate host-microbe interactions?
12
Q
What is genome editing and how can it be done?
A
It is introduction of a site-specific ds break
A:
- Ku heterodimers bind to DSB ends → recruit repair proteins
- Rad51 proteins bind DSB ends → recruit accessory factors → direct genomic recombination with homology arms
B:
- Zinc finger (ZF) proteins and transcription activator-like effectors (TALEs) are naturally occurring DNA-binding domains
- ZF and TALE domains each recognize 3 and 1 bp of DNA, respectively
- ZF or TALE domains can be fused to FokI endonuclease
C:
Type II CRISPR system can be engineered to be applicable in genome editing
13
Q
What are ZF-Nucleases?
A
Zinc finger (ZF) proteins
- Cys2-His2 zinc finger domain contacts 3bp of sequence in major groove
- Can be used as modular component to get sequence specific targeting of Fokl restriction endonuclease monomer. Cleavage requires targeting second monomer to other strand to generate functional Fokl dimer.
14
Q
What are TALENs?
A
Transcription activator-like effectors (TALEs)
- TALE effector proteins secreted by Xanthomonas bacteria in order to activate host plant gene expression that aids infection.
- Modular composition
- Each repeat unit of 33-35 amino acids specifies one target base.
- TALENs are built with 18 repeats
- Can be used to construct designer Transcription Activator Like Effector Nuclease (TALEN) to introduce DNA breaks at defined target sequence
15
Q
How can CRISPR-Cas be engineered?
A
fusion between a crRNA and part of the tracrRNA sequence
→ single guide RNA (sgRNA)
- designed sgRNA library targeting 18’080 genes (3-4 sgRNA/gene)
- Transfect cells → select → screen for desired phenotype
