CRISPR-Cas9 gene editing uses guide RNA (gRNA) to direct Cas9 nuclease to specific genomic loci. The molecular requirement that limits Cas9 cutting to the correct target site (preventing off-target editing) includes:

• A A protospacer adjacent motif (PAM sequence, 5'-NGG-3' for SpCas9) immediately 3' of the target DNA sequence ✓
• B A 5' cap on the guide RNA that interacts with PAM sequences on target DNA
• C Phosphorylation of Cas9 by ATM kinase to activate its nuclease domains
• D Simultaneous binding of two guide RNAs flanking the target for Cas9 activation

Explanation

SpCas9 (from S. pyogenes) requires both: (1) complementary base-pairing between the 20-nt spacer in gRNA and the target protospacer DNA strand, and (2) a PAM sequence (5'-NGG-3') immediately 3' of the protospacer on the non-template strand. The PAM is required for R-loop formation and Cas9 conformational activation; its absence prevents cutting even with perfect gRNA complementarity. This PAM requirement restricts targetable sites but also constrains off-target cutting. High-fidelity Cas9 variants further reduce off-target activity via weakened non-specific DNA contacts.

Reference: Harper's Illustrated Biochemistry, 32nd ed.

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

Written and medically reviewed by the StethoPrep medical team.