In CRISPR-Cas9, what sequence is essential for Cas9 to recognize the target?
Streptococcus pyogenes Cas9 requires a protospacer adjacent motif PAM downstream of target for binding and cleavage. The canonical PAM is 5'-NGG-3' on non-target strand. Guide RNA pairs with 20 nucleotides upstream of PAM, but Cas9 first scans DNA for PAM, triggering DNA unwinding and R-loop formation. Without PAM, even perfect guide complementarity fails to induce double strand break. PAM ensures self versus non-self discrimination since bacterial CRISPR array lacks PAM. Engineering higher fidelity Cas9 variants often alters PAM recognition profiles.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
