CRISPR-Cas9 genome editing uses a single guide RNA (sgRNA) that base-pairs with the target DNA. The Cas9 nuclease requires a PAM (protospacer adjacent motif) sequence on the non-template strand for target recognition. The most widely used Streptococcus pyogenes Cas9 uses which PAM sequence?

• A 5'-NGG-3' immediately 3' of the protospacer on the non-template strand ✓
• B 5'-NGA-3' immediately 5' of the protospacer on the template strand
• C 5'-NNGRRT-3' (R = purine) immediately 3' of the protospacer
• D 5'-TTN-3' immediately 5' of the protospacer on the non-template strand

Explanation

Streptococcus pyogenes Cas9 (SpCas9), the most widely used CRISPR nuclease, requires the PAM sequence 5'-NGG-3' (N = any nucleotide, G = guanine) located immediately 3' of the 20-nt protospacer on the non-template strand (the strand matching the sgRNA). PAM recognition by the PAM-interacting (PI) domain of Cas9 is the initial step that triggers R-loop formation and strand separation. Without PAM, Cas9 cannot bind stably to target DNA regardless of sgRNA complementarity. SaCas9 (Staphylococcus aureus) uses 5'-NNGRRT-3' PAM. CjCas9 (Campylobacter jejuni) uses a longer PAM. The PAM requirement defines the targetable space in the genome and is a critical consideration when designing sgRNAs, since targets lacking an appropriate PAM cannot be edited with SpCas9.

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

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

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