Lecture 1 - CRISPR I Flashcards
Define the 3 Domains of Life
- Bacteria
- Archaea (separated due to Sequencing technology)
- Eukarya (May have evolved from Archaea)
How are Microbial Communities Characterised?
(3 Points)
- DNA extraction, and either:
1. Amplicon Sequencing - multiple copies of fragments from 1 target gene
2. Metagenomics Sequencing - short sequences from all DNA
What are the Asgard Archaea?
(2 Points)
- Group (Superphylum) of deep-sea archaea found around hydrothermal vents
- Contain genes believed to be exclusive to eukaryotes, hence it has been suggested that eukaryotes may have evolved from this group
What is the function of CRISPR locus?
Provides acquired resistance to viruses in prokaryotes e.g., Bacteria
What are the key features/components of the CRISPR locus?
(3 Points)
- Leader Sequence - promoter for transcription of CRISPR loci
- Spacers - short segments of DNA captured from infecting MGEs
- Repeats
What is the role of Bacteria in yoghurt production?
Bacterial Lactase converts Lactose into Glucose and Galactose, which can be fermented by other bacterial enzymes into Lactic Acid
How did Bacteriophages contribute to the development of bacterial defence mechanisms?
Bacteriophage attacks are very common, providing a significant selection pressure that drives the evolution of defecnce mechanisms (e.g., CRISPR systems)
How was CRISPR Immunity Discovered?
(2 Points)
- Bacterial Cultures showed resistance to phage infection that corresponded with presence of CRISPR “Spacers” matching phage DNA sequences
- CRISPR Locus Expansion - correlated with increased resistance to specific bacteriophage species
Is CRISPR Immunity a form of Mendelian or Lamarckian Inheritance?
Lamarckian Inheritance - transformation by changes brought about via acquired characteristics (e.g., CRISPR spacers) that can be passed on to offspring
Define the two Classes of CRISPR Systems, explaining the difference between them
(2 Points)
- Class I (Types I, III, IV) - 95% of systems, utilise multi-subunit effector complexes comprised of several Cas proteins
- Class II (Types II, V, VI) - 5% of systems, utilise a single, large multidomain effector protein
How does CRISPR RNA (crRNA) production/processing vary between Class I and II Systems?
(2 Points)
- Class I - crRNA is produced by dedicated complex of multiple Cas proteins
- Class II - crRNA is processed either by Rnase III with help of transacting CRISPR RNA (tracrRNA), or by effector protein involved in target cleavage
(i) Define CRISPR Adaptation
(ii) What does it Require
(i) Capture and Integration of MGE spacers
(ii) Requires the Cas1/Cas2 proteins in all CRISPR types
Describe the structure of the Cas1/2 DNA Capture Complex?
o Hetero-multimer consisting of:
* One Cas2 Dimer
* Two Cas1 Dimers
Describe the DNA Capture Mechanism
Cas1-Cas2 Complex is loaded with a protospacer due to two of the Cas1 monomers active sites recognising the PAM in the short 3’ ssDNA flayed ends
(i) Describe the structure of the Protospacer
(ii) Why does the 23bp duplex have flayed ssDNA ends?
(i) 23bp DNA duplex with short 1-3nt ssDNA flayed ends
(ii) dsDNA ends are splayed due to tyrosine wedges in each Cas1 dimer, which lock open DNA branch points whilst fixing in place 23bp dsDNA
How is the Spacer Length Determined?
Governed by fixed disance between two Cas1 wedges, with the Cas2 dimer serving to hold active sites of Cas1 at exact length to produce correct sized spacer
What is the Role of Integration Host Factor (IHF) in CRISPR Spacer Integration?
- Binds to Leader Sequence of CRISPR array, distorting DNA (180º bend)
- Cas1 interacts with IHF to localise spacer integration immediately downstream of Leader (Leader-Repeat 1 Boundary) via nucleophilic attack
Describe Naïve Adaptation
Generation of Immunity for first time against MGE that has not been previously encountered (relatively unknown process)
Describe Primed Adaptation
(2 Points)
- Interference Complexes cut up MGE, forming DNA fragments that can be captured by Cas1/2 to form new spacers (update/extend pre-existing immunity)
- Cas1/Cas2 complex - physically/functionally interacts with interference complex, guiding it to MGE DNA for Capture (PAC - Primed Adaptation Complex)
