Below is the sequence of the HEXA gene with the underlined portion the 1278TATC allele that recessively caused Tay-Sachs disease. Answer the following questions about this sequence: 5’ 3’

3’ 5’

a. Identify all the PAM sites by clearly marking them in the sequence above.
b. Pick one that is appropriate for the goal of removing the 4 extra TATC nucleotides. There are multiple good answers and you should only choose one.
c. Design a 20bp sgRNA spacer sequence that will make an appropriate cut. There are multiple good answers, and you should only choose one. Be sure to label 5’ and 3’ and the side that would attach to the scaffold sequence.
d. Would you use an HDR template or NHEJ to consistently make the correct edit? Explain