CRISPR

Question 1

Where is CRISPR naturally found?

Answer 1

About 50% of bacteria and 75% of archaea

Question 2

What are the two types of repeating units found in the CRISPR locus?

Answer 2

Repeats and spacers, each about 24-28 bp long

Question 3

What are the neighbouring protein encoding genes beside the CRISPR locus? What do they do?

Answer 3

Cas genes. The proteins will bind to the sequence that corresponds to one of the spacers and does the cutting

Question 4

Where do the spacers come from?

Answer 4

Viral DNA that the bacterial cell fought off

Question 5

How does adaptive immunity work in bacteria using CRISPR?

Answer 5

1. Short sequences of foreign viral DNA are inserted into the CRISPR locus as a spacer
2. The entire CRISPR locus is transcribed into pre-crRNA, then processed into short crRNA made of one repeat and one spacer
3. The crRNA recognizes and binds to the DNA of the same bacteriophage that tried to infect it before, recruits Cas proteins which cut the viral DNA and destroys it

Question 6

How is CRISPR different from restriction enzymes?

Answer 6

Restriction enzymes will always cut the exact same sequence no matter what, but CRISPR can cut anything depending on what it gets for guide RNA

Question 7

How do cells normally fix a double strand break? What problems does this mechanism often have?

Answer 7

Non homologous end joining mechanisms. It is error prone and uses the homologous chromosome as a guide, so it often adds small indels

Question 8

How does CRISPR work as a lab technique?

Answer 8

1. The guide RNA binds to the target DNA sequence
2. Cas9 makes a double strand break
3. A piece of donor DNA is supplied, and the repair machinery thinks it’s a homologous chromosome and uses it as a template to repair the strand break
4. The cell repair machinery repairs the strand break to look like the donor DNA

Question 9

What are 3 advantages of CRISPR as a technique?

Answer 9

1. The recognition sequence of CRISPR is about 25 nucleotides long, so the odds of that sequence occurring anywhere else by chance is very low
2. Can be used in intact cells and organisms
3. Can modify many genes at the same time

Question 10

What is a disadvantage of CRISPR as a technique?

Answer 10

Off-target effects are possible. The guide RNA can bind to and end up modifying somewhere else in the genome that we don’t want

Question 11

How can CRISPR be applied to food production?

Answer 11

Creating virus resistant bacteria for yogurt and cheese production

Question 12

How can CRISPR be applied to basic research?

Answer 12

Can create a mutation that targets one particular gene to study what that gene does

Question 13

How can CRISPR be applied to agriculture?

Answer 13

Gene-edited crops and animals to improve various traits

Question 14

How can CRISPR be applied to medicine?

Answer 14

Can repair a disease causing mutation in a patient, repair mutations in tumours to stop growth, repair mutations for patients with genetic disorders