Genome Engineering

Question 1

What is CRISPR/Cas9?

Answer 1

A technique that uses a guide RNA to direct the Cas9 protein to a specific location in the genome, allowing for precise editing of DNA

Question 2

What are TALENs?

Answer 2

Transcription activator-like effector nucleases

Question 3

What are ZFNs?

Answer 3

Zinc factor nucleases

Question 4

What can CRISPR/Cas9 produce?

Answer 4

Because it functions in trans, plants can be generated that have no GE construct present

Question 5

What are the EU laws about gene editing?

Answer 5

GMO means organisms in which genetic material has been altered in any way that does not occur naturally by mating / natural recombination
However, 2018, it was ruled that CRISPR derived plants would be classified as GMO, even though the foreign construct could be outcrossed from the final product
2023, UK ruled CRISPR can be used for crops as long as there is no GM product in the end material.

Question 6

What does CRISPR stand for?

Answer 6

Clustered regularly interspaced short palindromic repeats

Question 7

What are the 2 pathways to repair double stranded breaks?

Answer 7

• Non homologous end joining: NHEJ, can introduce deletions or insertions to make sure break is joined
• Homology directed repair: HDR, template required to repair (oligo, a small single stranded DNA which contain point mutations)

Question 8

What are the 4 main components that CRISPR relies on?

Answer 8

1. Cas9 - endonuclease, with two nuclease domains (RuvC, HNH), each cleaves one strand of the DNA duplex
2. crRNA - RNA that targets the DNA, has a high sequence identity (20 nts)
3. tracrRNA - binds to crRNA and recruits Cas9 to the recognition site
   ** crRNA and tracrRNA can be fused into a single molecule called guide RNA (gRNA)
4. PAM sequence (NGG), gRNA must be adjacent to this

Question 9

What is a growing requirement for CRISPR/Cas9 vector systems?

Answer 9

Cas9 and gRNA cassettes to be separated to prevent them acting as a gene drive

Question 10

What promoters must Cas9 and gRNA have?

Answer 10

Cas9 - Pol II promoter and a transcriptional terminator to prevent read through
gRNA - Pol III promoter, not polyadenylated or capped, initiate and terminate in exact positions

Question 11

What is multiplexing?

Answer 11

gRNAs targeting multiple genes at the same time

Question 12

What are the two strategies for expressing multiple gRNAs?

Answer 12

1. Each gRNA has its own pol III promoter, drives the transcription of gRNAs separately. Each gRNA includes a sequence specific region and a tracrRNA region
2. Multiple gRNAs transcribed as one long RNA molecule from a single promoter. Endogenous tRNA processing enzymes cleave tRNA at specific sites, releasing individual gRNAs from the polycistronic strand

Question 13

What is CRISPR outcome?

Answer 13

CRISPR outcome is not predictable
Diploids have 2 alleles, GE could generate 2 different alleles in the same cell
Must be separates and then crossed to generate homoplasy

Question 14

What has CRISPR engineering done to tomatoes?

Answer 14

• Aim: combine differing fruit size with branching variations
  Reducing CLV3 increases WUS expression, knocking it out produces larger fruit
  Using CRISPR, promoter was targeted rather than the protein, however this produces 2 alleles in the same cell, so must wait until the second generation