Question

You are interested in using CRISPR to edit a particular gene in the genome. Select the correct order of events that must occur to introduce a double stranded DNA break using CRISPR/Cas9.

1. small guide RNA (sgRNA) hybridizes with the target DNA
2. Cas9 cuts DNA
3. Cas9/sgRNA complex binds PAM sequence
4. sgRNA binds Cas9
5. Cas9 unzips DNA

Answer 1

4. sgRNA binds Cas9

3. Cas9/sgRNA complex binds PAM

5. Cas9 unzips DNA

1. small guide RNA (sgRNA) hybridizes with the target DNA

2. Cas9 cuts DNA

Step-by-step explanation:

Background Knowledge:

Every cell in living body have genome (DNA). DNA is double helical structure which is held together by complementary base pairing rule i.e Adenine always form pair with Thymine and Guanine always pair with cytosine. Researchers have identified thousand of genes that can cause disease conditions in our body. Changing these genes in our body is not easy but in today's world it is possible through technique CRISPR.

CRISPR:

This method is based on natural system used by bacteria against the DNA of bacteriophages by activating CAS9 protein which due to guide RNA recognizes the target DNA and cut it into pieces.

Mechanism of CRISPR:

• Initially RNA called small guide RNA form complex with CAS9 endonuclease protein.
• This produces Cas9/sgRNA complex which then binds PAM sequence present inside target DNA.
• On binding with PAM sequence Cas9 unzips DNA.
• Small guide RNA (sgRNA) which is present in CAS9 complex hybridizes with the target DNA.
• This hybridization is identified by Cas9 which cuts the target DNA.