CRISPR-Cas9 - In Bacteria

Question 1

What does CRISPR stand for?

Answer 1

Cluster Regularly Interspaced Short Palindromic Repeats

Not needed to memorise

Question 2

Describe CRISPR based on how it looks.

Answer 2

• CRISPR is a big gene that begins with DNA that codes for Cas proteins.
• Then there are repeated palindromic sequences with spacers found between them

Question 3

What is Cas-9?

Answer 3

An endonuclease assosciated with CRISPR

Question 4

What is the role of CRISPR-Cas9 in bacteria?

Answer 4

• To protect against bacteriophage infection by being a form of adaptive immunity

Question 5

What are the 3 steps to fighting a Bacteriophage infection with bacteria using CRISPR-Cas9?

Answer 5

1. Exposure
2. Expression
3. Extermination

Question 6

What is the 1st step in Exposure for CRISPR-Cas9?

What invades?

Answer 6

• Bacteriophage injects viral DNA into bacteria.

Question 7

What is the 2nd step in Exposure for CRISPR-Cas9?

Cas1 and Cas2

Answer 7

• Cas1 and Cas2 (Two other endonucleases assosciated with CRISPR) cut out short section of viral DNA upstream to PAM.
• This is known as protospacer

Vut piece is about 30 nucleotides long

Question 8

What is 3rd step in Exposure for CRISPR-Cas9?

CRISPR gene

Answer 8

• Protospacer is added to bacteria’s CRISPR array.
• This becomes a spacer
• Each spacer is separated by a palindromic sequence of DNA

Question 9

What is a palindrome?

Answer 9

Something that can be read from either end.
Eg: Race car

Question 10

What is 1st step in Expression for CRISPR-Cas9?

Transcription

Answer 10

• CRISPR spacers are each transcribe with half of adjacent palindromic sequence on one side of spacer
• A guide RNA (gRNA) molecule is created

Question 11

What is 2nd step in Expression for CRISPR-Cas9?

Complex

Answer 11

• gRNA binds to Cas9 protein to create CRISPR-Cas9 complex
• Cas-9 protein can now target any Viral DNA with same base sequence as gRNA

Question 12

What is the 1st step in Extermination for CRISPR-Cas9?

Searching

Answer 12

• CRISPR-Cas9 complex searches for PAM sequence in all DNA present in cell

Question 13

What is PAM sequence?

Answer 13

• Protospacer Adjacent Motif
• A short DNA sequence (2-6 bases long)
• Found adjacent to DNA region of spacer

Question 14

Why is PAM important for Cas-9?

Answer 14

It is required to be present for Cas9 to cut DNA. Cas-9 will not cut DNA without PAM being present.

Question 15

What is 2nd step in Extermination for CRISPR-Cas9?

Matching sequence

Answer 15

• Once PAM is found, DNA is unwound and separated
• Then, Cas-9 checks to see if DNA near PAM is complementary to gRNA
• If not complementary, DNA is rewound and Cas9 continues earch for more PAMs

Question 16

What is 3rd step in Extermination for CRISPR-Cas9?

Match found

Answer 16

• CRISPR-Cas9 cleaves the phosphate-sugar backbone of both DNA strands.
• It can cut both strands as it has 2 active sites.
• Blunt ends are created.

Question 17

What is 4th step in Extermination for CRISPR-Cas9?

Aftermath

Answer 17

As a result, foreign Bacteriophage DNA is destroyed. Virus cannot reproduce or synthesis any proteins. Bacteria is protected.

Question 18

Describe how Cas9 will never cuts the gRNA sequence found on the CRISPR gene of bacterial DNA

Answer 18

As Bacterial DNA’s CRISPR gene does not contain PAM sequence, only Viral DNA does