Genome editing

Question 1

What are the 3 broad classes of genome editing nucleases?

Answer 1

1. Zinc finger nucleases
2. TALENs
3. CRISPR

Question 2

What is the zinc finger nuclease?

Answer 2

A protein consisting of a DNA-cutting enzyme and a DNA-grabbing region that can be tailored to recognise different genes

Question 3

What are TALENs?

Answer 3

A protein containing a DNA-cutting enzyme and a DNA-grabbing region that can be programmed to recognise different genes, but it is easier to design than zinc finger nucleases

Question 4

What is CRISPR?

Answer 4

A DNA-cutting protein guided by an RNA molecule that is able to find the specific gene of interest

Question 5

How do zinc finger proteins work?

Answer 5

• Need to be individually designed to recognise a specific DNA sequence within the genome
• They bind to that sequence and cut it

Question 6

How do TALENs work?

Answer 6

-Have to be designed for a specific DNA sequence

Question 7

How does CRISPR work?

Answer 7

• Has specificity that is conferred by the guide RNA

- The nuclease which cuts the DNA is Cas9 and will work on any sequence of guide RNA as long as trace RNA remains there

Question 8

What does CRISPR stand for?

Answer 8

-CRISPR stands for clustered interspersed short palindromic repeats

Question 9

What is CRISPR-Cas9 based on?

Answer 9

-Based on bacterial adaptive immune system

Question 10

What can CRISPR-Cas9 be used for?

Answer 10

-Can be used for a wide variety of species and cells in culture

Question 11

What does CRISPR-Cas9 require?

Answer 11

-Requires a guide RNA with a protospacer adjacent Motif

Question 12

Steps involved in the CRISPR-Cas9 system

Answer 12

• We’d make a single guide RNA (sgRNA) which consists of a target specific crRNA sequence attached to a tracrRNA
• This sgRNA will be added into a Cas9 protein
• This will then induce the double stranded cleavage of the genomic DNA at a specific location
• Then the cellular machinery will try repair the double stranded break by non homologous end joining(NHEJ) or homology-directed repair(HDR)
  
  • Imprecise NHEJ mediated repair can produce insertion and/or deletion mutations of variable length at the site of the double stranded break
    
    • HDR mediated repair can introduce precise point mutations or insertions from a single stranded or double stranded DNA donor template

Question 13

Why are there CRISPR variations?

Answer 13

CRISPR-Cas9 has off target effects

Question 14

What is an example of a CRISPR variation method?

Answer 14

Genome engineering by double nicking with paired Cas9 nickases

Question 15

How does Genome engineering by double nicking with paired Cas9 nickases work?

Answer 15

• Cas9 has been modified so it has nickase activity
  
  • This means that it cleaves only one strand of genomic DNA and not both
• By targeting 2 DNA nearby, you can have 2 specific events and can then use HDR