How does CRISPR-Cas9 achieve targeted genome editing and what is the role of the PAM sequence?

How does CRISPR-Cas9 achieve targeted genome editing and what is the role of the PAM sequence?

• A. A guide RNA directs Cas9 to a complementary DNA sequence adjacent to a PAM; Cas9 introduces a double-strand break.
• B. Cas9 binds DNA without guide RNA and cuts at random.
• C. The PAM sequence is located within the guide RNA.
• D. Cas9 only makes single-strand nicks and does not require a PAM.

Answer: (C)

The essential idea is that targeted genome editing with CRISPR-Cas9 relies on two things: a guide RNA that carries the sequence matching the desired DNA target, and a PAM sequence on the DNA that Cas9 must recognize to cut. The guide RNA pairs with a complementary DNA sequence, guiding the Cas9 enzyme to that specific location. But Cas9 won’t cut unless the adjacent DNA contains the PAM motif, a short sequence standing next to the target site. This PAM check ensures the enzyme cuts only at the intended site and helps prevent unintended activity elsewhere in the genome. When both the guide RNA–DNA pairing and the PAM are present, Cas9 makes a double-strand break just upstream of the PAM. The cell then repairs that break through non-homologous end joining or homology-directed repair, enabling mutations or the insertion of new genetic material. The PAM lives on the DNA, not in the guide RNA, and its presence is what makes the targeting specific and controllable.

The essential idea is that targeted genome editing with CRISPR-Cas9 relies on two things: a guide RNA that carries the sequence matching the desired DNA target, and a PAM sequence on the DNA that Cas9 must recognize to cut. The guide RNA pairs with a complementary DNA sequence, guiding the Cas9 enzyme to that specific location. But Cas9 won’t cut unless the adjacent DNA contains the PAM motif, a short sequence standing next to the target site. This PAM check ensures the enzyme cuts only at the intended site and helps prevent unintended activity elsewhere in the genome. When both the guide RNA–DNA pairing and the PAM are present, Cas9 makes a double-strand break just upstream of the PAM. The cell then repairs that break through non-homologous end joining or homology-directed repair, enabling mutations or the insertion of new genetic material. The PAM lives on the DNA, not in the guide RNA, and its presence is what makes the targeting specific and controllable.