If the PAM sequence is mutated, what is the likely effect on Cas9-mediated cutting?

If the PAM sequence is mutated, what is the likely effect on Cas9-mediated cutting?

• A. Cas9 binding and cutting are greatly reduced or abolished.
• B. Cas9 binds more strongly.
• C. PAM mutation has no effect.
• D. Cas9 changes to target RNA.

Answer: (A)

The main idea is that PAM recognition is essential for Cas9 to engage and cut DNA. Cas9 relies on a short PAM sequence adjacent to the target site to initiate binding and activate its nuclease activity. If the PAM is mutated, Cas9 can no longer recognize the DNA correctly, so binding is greatly reduced or abolished and the double-strand break does not occur. That’s why mutating the PAM typically eliminates Cas9-mediated cutting. The other possibilities don’t fit because Cas9’s ability to bind and cleave depends on the presence of the correct PAM, and Cas9 is not used to cut RNA in its standard form.

The main idea is that PAM recognition is essential for Cas9 to engage and cut DNA. Cas9 relies on a short PAM sequence adjacent to the target site to initiate binding and activate its nuclease activity. If the PAM is mutated, Cas9 can no longer recognize the DNA correctly, so binding is greatly reduced or abolished and the double-strand break does not occur. That’s why mutating the PAM typically eliminates Cas9-mediated cutting. The other possibilities don’t fit because Cas9’s ability to bind and cleave depends on the presence of the correct PAM, and Cas9 is not used to cut RNA in its standard form.