Lecture 1 - CRISPR I

Question 1

Define the 3 Domains of Life

Answer 1

1. Bacteria
2. Archaea (separated due to Sequencing technology)
3. Eukarya (May have evolved from Archaea)

Question 2

How are Microbial Communities Characterised?

(3 Points)

Answer 2

• DNA extraction, and either:
  1. Amplicon Sequencing - multiple copies of fragments from 1 target gene
  2. Metagenomics Sequencing - short sequences from all DNA

Question 3

What are the Asgard Archaea?

(2 Points)

Answer 3

• 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

Question 4

What is the function of CRISPR locus?

Answer 4

Provides acquired resistance to viruses in prokaryotes e.g., Bacteria

Question 5

What are the key features/components of the CRISPR locus?

(3 Points)

Answer 5

1. Leader Sequence - promoter for transcription of CRISPR loci
2. Spacers - short segments of DNA captured from infecting MGEs
3. Repeats

Question 6

What is the role of Bacteria in yoghurt production?

Answer 6

Bacterial Lactase converts Lactose into Glucose and Galactose, which can be fermented by other bacterial enzymes into Lactic Acid

Question 7

How did Bacteriophages contribute to the development of bacterial defence mechanisms?

Answer 7

Bacteriophage attacks are very common, providing a significant selection pressure that drives the evolution of defecnce mechanisms (e.g., CRISPR systems)

Question 8

How was CRISPR Immunity Discovered?

(2 Points)

Answer 8

• 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

Question 9

Is CRISPR Immunity a form of Mendelian or Lamarckian Inheritance?

Answer 9

Lamarckian Inheritance - transformation by changes brought about via acquired characteristics (e.g., CRISPR spacers) that can be passed on to offspring

Question 10

Define the two Classes of CRISPR Systems, explaining the difference between them

(2 Points)

Answer 10

• 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

Question 11

How does CRISPR RNA (crRNA) production/processing vary between Class I and II Systems?

(2 Points)

Answer 11

• 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

Question 12

(i) Define CRISPR Adaptation

(ii) What does it Require

Answer 12

(i) Capture and Integration of MGE spacers

(ii) Requires the Cas1/Cas2 proteins in all CRISPR types

Question 13

Describe the structure of the Cas1/2 DNA Capture Complex?

Answer 13

o Hetero-multimer consisting of:
* One Cas2 Dimer
* Two Cas1 Dimers

Question 14

Describe the DNA Capture Mechanism

Answer 14

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

Question 15

(i) Describe the structure of the Protospacer
(ii) Why does the 23bp duplex have flayed ssDNA ends?

Answer 15

(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

Question 16

How is the Spacer Length Determined?

Answer 16

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

Question 17

What is the Role of Integration Host Factor (IHF) in CRISPR Spacer Integration?

Answer 17

1. Binds to Leader Sequence of CRISPR array, distorting DNA (180º bend)
2. Cas1 interacts with IHF to localise spacer integration immediately downstream of Leader (Leader-Repeat 1 Boundary) via nucleophilic attack

Question 18

Describe Naïve Adaptation

Answer 18

Generation of Immunity for first time against MGE that has not been previously encountered (relatively unknown process)

Question 19

Describe Primed Adaptation

(2 Points)

Answer 19

• 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)