CRISPR

Question 1

What does cas mean?

Answer 1

CRISPR-associated

Question 2

What is the purpose of cas genes?

Answer 2

They encode enzymes that can cut DNA in viruses. Cas genes create cas proteins.

Question 3

What is the leader?

Answer 3

The region separating CAS genes and the CRISPR array

Question 4

What is the CRISPR locus?

Answer 4

A section of the genome in bacteria that contains cas genes, a leader, and a CRISPR array containing spacers, and repeats

Question 5

What are spacer regions?

Answer 5

Regions in the CRISPR array that correspond to unique viral sequences. It’s the method through which bacteria recognize viruses and kill them; they keep data to recognize them in these spacers. They are variable in sequence.

Question 6

What are repeats?

Answer 6

They fall between spacers.

Question 7

What are cas proteins?

Answer 7

These are nucleases that bind and cleave viral DNA.
They detect when the virus has entered through enzyme bluetooth.

• They are nucelases/enzymes
• Recognize
• Bind
• Cleave

Question 8

How does a viral sequence turn into viral death?

Answer 8

A new spacer is derived from the virus and integrated as a spacer into the CRISPR array.

Transcription occurs and CRISPR RNA is formed.

CRISPR RNA is used to mobilize the molecular machinery that will cleave the viral genome. The cas proteins contained the processed the guide RNA. Guide RNA recognizes where to edit and then tells the cas protein the sequence to cut.

Question 9

What is cas 9 enzyme?

Answer 9

It is an endonuclease that cuts both DNA strands at a specific site and include heilcase activity

Question 10

What is single guide RNA?

Answer 10

An engineered RNA that forms a complex with Cas 9 composed of two parts.

It has a guiding region and a scaffold region. The scaffold region hooks onto the cas 9 protein and the guiding region is complementary to the DNA site of interest and functions as a spacer.

Question 11

What does the cas 9 complex contain?

Answer 11

A guiding region and a scaffold region.

Question 12

What is the guiding region?

Answer 12

It functions as a spacer and is complementary to the target region of DNA. Scientists customize this region for their research.

Question 13

What is the scaffold region?

Answer 13

This is the part of the sgRNA that hooks onto the cas 9 enzyme and attaches the sgRNA to it. It has hairpin loops for grip; it forms a multi-hairpin look struction that binds in a crevice of the cas9 protein.

Question 14

What is the PAM sequence?

Answer 14

The Protospacer Adjascent Motif. It is required for cas 9 to function because cas 9 recognizes the sequence, binds there, and seperates the DNA strands.

Question 15

What are the two ways the cut DNA can be repaired?

Answer 15

Non-homologous end joining or Homologous Directed Repair.

Question 16

What is NHEJ?

Answer 16

Enzymes rejoin the severed DNA strands either by adding in new bases or deleting ones. This can lead to mutations, and disrupted gene expression or function. We glue the strand back together.

Question 17

What is HDR?

Answer 17

Enzymes fix the double stranded break using template DNA. A complementary DNA strand is created. This process is used to generate precise genome changes.

Question 18

What is donor template DNA?

Answer 18

It is provided by the people conducting the lab, and it includes homology arms that match DNA sequences around the cut sit.

Question 19

What is lacZ?

Answer 19

A gene that encodes beta-galactosidase in E. coli

Question 20

What is beta-galactosidase?

Answer 20

An enzyme that breaks down lactose (substrate) and turns it into glucose. It can also break down x-gal.

Question 21

What is x-gal?

Answer 21

A substrate that, when broken down, produces a blue pigment that we can use to track gene expression.

Question 22

What is IPTG?

Answer 22

A lactose analog that activates the expression of lacZ. It is not consumed by the bacteria so it induces lacZ expression.

When there is enough lactose in the bacteria, to avoid toxic levels of it or waste energy producing more than necessary, lactose binds to a repressor enzyme that opens the doors to the enzymes like beta-galactosidose that can break the lactose down into glucose, etc. However, once the lactose is broken down, there is no more lactose to disactivate the repressor protein so it goes back to repressing those enzymes. IPTG allows us to keep the repressor inactive, let the enzymes out, and keep them out because they do not break down the IPTG.

Question 23

What is a codon?

Answer 23

A set of three nucleotides that correspond to a specific amino acid.

Question 24

What is a stop codon?

Answer 24

It tells the ribosomes to stop making proteins - to stop making the enzyme in this case (beta-galactosidose).

Question 25

How can we replicate and use this adaptive immunity in a lab?

Answer 25

We can direct the cutting of the double-stranded DNA and fix the break in a way that modifies the genetic sequence that has been cut. We modify the guide RNA in this system to be able to target a specific location of interest in the genome to cut a gene and see what happens, or cut a gene and change the base sequence at that location to generate precise genome changes.

Question 26

What is the point of the CRISPR lab/Blue-White Screening?

Answer 26

It tells us if our genome was successfully edited. If so, and we cut the lacZ up, it should now work anymore; it should not be able to produce functional beta-galactosidase and turn the substrate into a blue pigment. White is good. If it does turn blue, the enzyme is functional because it broke down the substrate, which means the gene was intact and working correctly, which means it did not change, which means we did not edit it successfully.

Question 27

What is a promoter?

Answer 27

A promoter that defines where transcription of a gene by RNA polymerase begins.

Question 28

What is arabinose?

Answer 28

A sugar like lactose that, when introduced to the bacterial cell, opens the door for enzymes to come out and break it down. In our lab, these enzymes enable DNA repair. Without them, the bacteria dies.

Question 29

What is in the donor plasmid?

Answer 29

It contains the donor DNA template but not the sgRNA. So, no cutting takes place and LacZ is expressed as normal both in the presence of arabinose and without it.

Question 30

What is the donor guide plasmid?

Answer 30

This one contains the donor guide template DNA along with the sgRNA which can bind to the cas-9 in all the bacteria and cut LacZ. In the presence of arabinose, the cut will be repaired and a stop codon will be introduced. This means the bacteria will reproduce without the expression of the LacZ gene, and without the blue pigment as a result. The gene editing has worked!

Without arabinose, the donor dna template cannot be used to repair the DNA - the machinery is not available. Therefore, with no NHEJ and a cut in the bacteria’s DNA, the cell dies.