CRISPR-Cas9 genome editing requires a guide RNA (gRNA) to direct Cas9 to the target sequence. The ESSENTIAL genomic sequence feature that determines permissible Cas9 cut sites adjacent to any target is:
- A A TATA box upstream of the target sequence for Cas9 loading
- B A CpG island within 100 bp of the target for stable Cas9 binding
- C A protospacer adjacent motif (PAM) sequence (5'-NGG-3' for SpCas9) immediately downstream of the target ✓
- D A consensus kozak sequence for Cas9 recognition of the correct reading frame
Explanation
SpCas9 (from Streptococcus pyogenes) requires a PAM (protospacer adjacent motif) sequence of 5'-NGG-3' immediately 3' of the target DNA sequence for Cas9 binding and cleavage. The PAM is recognized by the PAM-interacting (PI) domain of Cas9 and is NOT part of the gRNA sequence. Without a PAM, Cas9 cannot unwind the double-stranded DNA for gRNA hybridization. This PAM requirement limits targetable sequences to ~1 per 8 bp on average in mammalian genomes. Different Cas9 orthologs (SaCas9, CjCas9) and engineered variants (xCas9, SpCas9-NG) have altered PAM requirements to expand targeting flexibility.
Reference: Harper's Illustrated Biochemistry, 32nd ed.
High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP
Written and medically reviewed by the StethoPrep medical team.