Lecture 4 - CRISPR Systems in Antibiotic Development

Question 1

why is the CRISPR-Cas system used in bacteria?

Answer 1

protects bacteria from bacteriophages

Question 2

how does CRISPR-Cas system work? (5)

Answer 2

1. bacteriophage injects genome
2. Cas1 and Cas2 insert segment of phage DNA into bacterial DNA with spacers
3. crRNA is made and held by Cas9 as a surveillance complex
4. complex moves around cytoplasm to find matching sequence
5. target recognition can only occur if there is PAM recognition site, which is sequence next to target

Question 3

how is CRISPR-Cas9 used to make KO?

Answer 3

• target gene has PAM next to it
• target-specific sgRNA has tracrRNA hairpin which is scaffold for nuclease protein
• sgRNA binds Cas9 –> complex scans and finds gene of interest
• nuclease cleaves target gene
• repair mechanism is error prone so gene is KO

Question 4

what are the 2 most common methods for gene modulation?

Answer 4

1. CRISPRi
2. CRISPRa

Question 5

how do CRISPRi and CRISPRa work?

Answer 5

Cas9 has mutation so it cannot cut

complex goes to promoter to block (CRISPRi) or overexpress (CRISPRa) gene

Question 6

3 ways of inducing gene mutation

Answer 6

1. Cas9 + HDR
2. Base editors
3. Prime editors

Question 7

how does Cas9 + HDR work?

Answer 7

tricks cell into thinking that DNA you add is part of a sister chromatid to be replicated from

Question 8

how can we use CRISPRi to look at M. Tb drug resistance?

Answer 8

block many target genes with CRISPRi
- if that gene is involved in resistance –> blocking = no resistance
- if that gene drives sensitivity to drug –> blocking = resistance

Question 9

describe volcano plot

Answer 9

X axis = log fold change
- >0 means KO of gene causes bacterial cells to grow better (RESISTANCE gene)
- <0 means KO of gene causes bacterial cells to die (SENSITIVITY gene)

Question 10

2 results of the CRISPRi screening

Answer 10

1. block mtrA (involved in cell wall) –> sensitivity
2. block bacA (transporter) –> resistance

Question 11

significance of blocking mtrA to cause drug sensitivity

Answer 11

if cell wall cannot remodel properly, the drug can more easily get in so the cell is more sensitive

Question 12

significance of blocking bacA to cause drug resistance

Answer 12

without this transporter, the drug cannot enter the cell as easily so the cell is resistant

Question 13

how can we use CRISPR system itself as an antibiotic?

Answer 13

use bacteriophage to deliver RNA into bacteria to target essential bacteria genes –> kill bacteria