Question

4. What is the function of each component of the CRISPR-Cas9 system? (1.5 pt) PAM site: Guide RNA: Cas9: 5a. In the DNA below, the "I" line indicates where there is a mutation in a gene. Design a gRNA to find this mutation. Your gRNA will be 8 bases long and will not include the PAM sequence. Keep the following in mind: [2 pts total] i. Scan the top strand left to right and find the PAM site. ii. When you find it, highlight it (1/2 pt] ill. Then do a U-turn and identify the 8-base Target Sequence associated with it. Underline it. (1/2 pt) iv. Then design the gRNA that will complementary base pair with it to guide the Cas9 enzyme to this location (1 pt) BRNA: GAGCTCTCATAGACAGGA IIIIIIIIIIIIIIIIII CTCGAGAGTATCT GTCCT 5b. Cas9 cuts at the point 3 bases to the left of the PAM site. In the DNA above, draw arrows pointing to the location where Cas9 will snip the DNA. (0.5 pt) 5c. If the mutation indicated by the two short vertical lines in the DNA is the site of a missing sequence of DNA, will the following repair DNA insert in the correct location? (0.5 pt] YES NO TAG ATCATTGACA IIIIIIIIIIIII ATC TAGTAA CTGT

Answer 1

4) The function of PAM site is - PAM is Protospacer Adjacent Motif and is a short DNA sequence of 2-6 base pairs. It follows the DNA sequence that is targeted for cleavage by the CRISPR system.This sequence is required by the Cas nuclease to make the cut. It also distinguishes self from non-self DNA in bacteria and hence prevents the wrong segment from being cut.

The function of guide RNA IS- Guide RNA is a specific RNA sequence which is about 100 base pairs in length. Its main function is to recognize the target DNA sequence which is to be cleaved and guides the Cas nuclease there for cleavage.

The main function of Cas9 is - Cas9 is a nuclease enzyme produced by the CRISPR system. Its main function is to bind to the DNA of interest and cleave it.