Lecture 12: CRISPR Flashcards

1
Q

What does CRISPR stand for?

A

Clustered Regularly Interspaced Short Palindromic Repeats

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

How does the RNA guided immune system work to protect bacteria from viral infection?

A
  1. When a bacterium encounters a new viral invader, it captures a small snippet of the viral DNA and incorporates it into its own genome within a region called the CRISPR array. These snippets are known as spacers.
  2. The CRISPR array is transcribed into a long RNA molecule called pre-crRNA (pre-cursor CRISPR RNA). This pre-crRNA is then processed into shorter crRNAs (CRISPR RNA) each containing a single spacer sequence along with a repeat sequence.
  3. The crRNAs then form complexes with Cas proteins (CRISPR-associated proteins), creating an RNA-guided surveillance complex. When the bacterium encounters the same viral invader again, the crRNA guides the Cas proteins to the viral DNA or RNA that matches the spacer sequence.
  4. Once the complex binds to the viral DNA or RNA, the Cas proteins cleave it, effectively neutralizing the viral threat. This prevents the virus from replicating and infecting the bacterium.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is the mechanism of action of siRNA drugs?

A
  1. ASGPR on the cell surface mediates the endocytosis of the siRNA into the cell
  2. Endosomal and lysosomal entrapment creates an intracellular drug depot
  3. The siRNA is slowly and continuously released into the cytoplasm over months
  4. The loading of siRNA onto a protein forms RNA-induced silencing complex (RISC)
  5. The passenger strand is removed, leaving only the guide RNA
  6. The guide RNA helps to locate the target mRNA and the protein cleaves it, which stops it from being translated
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What is the specific site where the cas9 protein cuts the DNA?

A

It is the PAM sequence that has to be at the 3’ end of the guide RNA, and the RNA itself should not contain this sequence

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is the protospacer adjacent motif (PAM)?

A
  1. PAM is a DNA sequence immediately following the DNA sequence targeted by the Cas 9 nuclease in the CRISPR bacterial adaptive immune system.
  2. PAM sequence: 5’-NGG-3’ for Cas9
  3. The cut happens 3 nucleotides upstream of PAM
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What happens if the gene you want to target doesn’t have the 5’-NGG-3’ PAM sequence?

A

Use another CAs system that requires other PAM sequences

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What is single guide RNA?

A
  1. It’s a specific RNA sequence that recognizes the target DNA region of interest.
  2. In order to cut, a specific sequence of DNA called PAM of between 2 and 5 nucleotides must lie at the 3’ end of the guide RNA
  3. Made up of 2 parts: crRNA and tracrRNA
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What is the function of tracrRNA in a CRISPR-Cas9 system?

A

It serves as a binding scaffold for the Cas nuclease

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What sequence should the guide RNA not contain and why?

A

It should not contain the PAM sequence otherwise the Cas 9 will cleave it

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is the difference between gRNA and sgRNA?

A

gRNA: guide RNA
sgRNA: single guide RNA
sgRNA contains both the crRNA and the tracrRNA fused together
gRNA: the sgRNA and tracrRNA components are separate

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What are the two types of repair that happen after CRISPR-CAs9 cut the DNA sequence?

A
  1. Non homologous end joining (NHEJ)
  2. Homology directed repair (HDR)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the issue with non homologous end joining?

A

Just to clarify, the issues with NHEJ make it good for CRISPR:
1. It’s error prone
2. Causes small insertions or deletions (indels) at the site of breakage
3. The randomness results in a diverse array of mutations
4. Ideally for CRISPR this will result in a loss of function mutation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Which repair mechanism is the most active one for DNA repair?

A

Non homologous end joining (NHEJ)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Which repair mechanism out of the 2: Non homologous end joining and Homology directed repair is more efficient and error prone?

A

Non homologous end joining: efficient but error prone.
Homology directed repair: non efficient but high fidelity.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is the entire process of carrying out CRISPR?

A
  1. Design guide RNAs and obtain the custom synthesized sgRNAs and the CAS9 enzyme
  2. Preassemble sgRNA and Cas9 into RNP complexes by mixing them in proper ratios and immediately transfect them into target cells by electroporation or with chemical reagents
  3. Grow without antibiotics for 4-5 days until 80–90% confluence.
  4. Perform initial screening with SURVEYOR assays or Sanger DNA sequencing of the gRNA target region
  5. Perform clonal selection by limited dilution of cells on a 96-well culture plate. Colonies appear after ~ 3–4 weeks
  6. Transfer colonies from 96-well plates to 6-well plates for expansion
  7. Perform phenotypic and genotypic characterization of individual clones
How well did you know this?
1
Not at all
2
3
4
5
Perfectly