Lecture 12: CRISPR

Question 1

What does CRISPR stand for?

Answer 1

Clustered Regularly Interspaced Short Palindromic Repeats

Question 2

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

Answer 2

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.

Question 3

What is the mechanism of action of siRNA drugs?

Answer 3

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

Question 4

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

Answer 4

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

Question 5

What is the protospacer adjacent motif (PAM)?

Answer 5

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

Question 6

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

Answer 6

Use another CAs system that requires other PAM sequences

Question 7

What is single guide RNA?

Answer 7

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

Question 8

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

Answer 8

It serves as a binding scaffold for the Cas nuclease

Question 9

What sequence should the guide RNA not contain and why?

Answer 9

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

Question 10

What is the difference between gRNA and sgRNA?

Answer 10

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

Question 11

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

Answer 11

1. Non homologous end joining (NHEJ)
2. Homology directed repair (HDR)

Question 12

What is the issue with non homologous end joining?

Answer 12

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

Question 13

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

Answer 13

Non homologous end joining (NHEJ)

Question 14

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

Answer 14

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

Question 15

What is the entire process of carrying out CRISPR?

Answer 15

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