Lecture 2 - CRISPR II

Question 1

Define Interference

Answer 1

Process of Surveillance, Targeting and Degradation of MGE DNA

Question 2

(i) How is crRNA processed and loaded into interference complexes
(ii) How does crRNA mediate interference?

Answer 2

(i) CRISPR loci is transcribed to RNA (pre-crRNA), which is processed and loaded into interference proteins
* crRNA - produced from single DNA spacer

(ii) crRNA in interference protein complexes base-pairs with complementary MGE DNA, targeting nucleolytic activity of interference complex to specific sequences

Question 3

Compare the interference complexes produced for Class I and Class II CRISPR Systems

Answer 3

Class I - multiprotein surveillance complex (cascade), which recruits nuclease-helicase Cas3 to cleave MGE

Class II - single protein complex, which may require tracrRNA for assembly (signals Cas9 to fold/activate)

Question 4

(i) How is the R-loop formation triggered/ activated?
(ii) What is this known as?

(3 Points, 1 Point)

Answer 4

(i) R-loop formation is triggered by two factors:
* Detection of PAM sequence
* Formation of seed region (locks R-loop in place)

(ii) Known as Surveillance Events of Interference

Question 5

How is the R-loop involved in the interference process?

Answer 5

R-loop formation recruits effector proteins (e.g., Cas3 - recognises ssDNA) that cleave up the MGE DNA

Question 6

Define the two major functions of Interference Protein Complexes

Answer 6

1. Surveillance for complementary MGEs
2. DNA cleavage to destroy recognised MGEs

Question 7

(i) What is the Seed Region?
(ii) Why is Seed Formation important?

Answer 7

(i) 6-8nt upstream of PAM sequence which must be complementary to the target for recognition
* provides specificity

(ii) Seed Formation (i.e. Base Pairing) - provides sufficient energy for “snapping/locking” onto DNA, forming the R-loop structure

Question 8

(i) What is the Function of the Protospacer Adjacent Motif (PAM)?

(ii) How do they functionally couple adaptation and interference?

Answer 8

(i) Marks site of protospacer sequence for interference complex, but is not integrated into CRISPR Loci to prevent self-recognition

(ii) Involved in both Interference and Adaptation

Question 9

What is the CasA/Cse1 subunit?

Answer 9

Component of cascade interference complex that recognises PAM

Question 10

(i) How does the CasA subunit recognise the PAM?
(ii) Why is this significant?

Answer 10

(i) PAM sequence (e.g., ATG) is recognised in double strand form via its minor groove (H-bond patterns)

(ii) This recognition method allows some mismatched PAMs to be tolerated, providing target strand sequence is optimal

Question 11

Define Cas3 in terms of:
(i) Structure + active site composition
(ii) Recruitment
(iii) Function

Answer 11

(i) Multidomain Polypeptide, containing HD domain in active site (Catalytic Histidine/Aspartate residues)

(ii) Recruited to Interference complex via ssDNA

(iii) ATP-dependent Translocase/Nuclease, moving along ssDNA and cleaving it into fragments

Question 12

Describe the Structure/Assembly of the Cas9 Interference complex

Answer 12

crRNA base-pairs with tracrRNA, which forms a scaffold that recruits/loads onto the Cas9 effector protein

Question 13

What are the two major functions of tracrRNA?

Answer 13

1. Cutting pre-crRNA into crRNA, comprising of a single spacer sequence (by RNase III)
2. Activation of Cas9 apoenzyme into haloenzyme/ nuclease enzyme

Question 14

Describe the structures of the two Cas9 Nuclease active sites

Answer 14

1. RuvC-like - based upon Aspartate residues
2. HNH/RNase-like - based on a Histidine residue

Question 15

How can Cas9 be used for genome editing?

(2 Points)

Answer 15

• Cas9 nuclease activity can be utilised to induce double-strand breaks at specific sites within the genome (due to crRNA/gRNA)
• dsDNA breaks activate DNA repair mechanisms, which can lead to gene KO, mutational insertion or repair of mutated genes

Question 16

(i) What was 1st Generation Cas9 Genome Editing?
(ii) What is it used for?

(1 Point, 2 Points)

Answer 16

(i) Targeting of WT-Cas9 enzymes to sites in the genome with a sgRNA, leading to dsDNA breaks and activation of DNA repair mechanisms (NHEJ/HR)

(i) Useful for studying effect of gene KO, less so for therapeutic benefit (to imprecise)
* dsDNA break repair is complex, and may generate unwanted genetic changes

Question 17

What is 2nd Generation Cas9 Genome Editing?

Answer 17

Genome editing process which avoids dsDNA breaks by utilising inactivated Cas9:

• “nickase” nCas9 - inactivated at one active site, hence only nicks one DNA strand
• “deactivated” dCas9 - inactivated at both active sites

Question 18

(i) What is CRISPR Prime Editing?

(ii) How does it work?

Answer 18

(i)Type of nCas9-based genetic modification,

(ii) Reverse transcriptase is fused to nCas9 C-terminus, and generates new DNA by copying RNA template in prime-editing guide RNA (pegRNA) that also functions as sgRNA

Question 19

What type of DNA Repair process is required for nCas9 Prime Editing?

Answer 19

Mismatch Repair - which repairs the mismatched bases in order to insert the edited sequence

Question 20

How can a DNA “Nick” on the unedited strand be produced?

(2 Points)

Answer 20

1. sgRNA + nCas9 alongside prime-editing complex
2. Host Mutational Proteins (e.g., MutSa/MutLa)

Question 21

How can nCas9 Prime Editing be utilised therapeutically?

Answer 21

May be used to treat certain monogenetic diseases (e.g., Tays-Sachs Disease)