CRISPR-Cas9 gene editing uses a guide RNA (gRNA) to direct Cas9 nuclease to cleave a target DNA sequence. A mandatory requirement for Cas9 cleavage adjacent to the target site is the presence of:

• A A PAM sequence (5'-NGG-3' for SpCas9) immediately 3' to the target in the non-template strand ✓
• B A TATA box promoter sequence upstream of the target
• C A polyadenylation signal 3' to the cut site
• D A CpG island within the target DNA sequence

Explanation

SpCas9 (from S. pyogenes) requires a protospacer adjacent motif (PAM) sequence 5'-NGG-3' immediately 3' of the target protospacer sequence in the non-template strand for DNA cleavage. The gRNA base-pairs with the complementary protospacer sequence, and PAM recognition is required for Cas9 to adopt its active endonuclease conformation and cleave both DNA strands (3 bp upstream of the PAM). This PAM requirement limits targeting flexibility (every NGG in the genome is a potential target). Alternative Cas proteins with different PAM requirements (e.g., SaCas9: 5'-NNGRRT-3'; AsCas12a: 5'-TTTV-3') have been developed to expand targeting.

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

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

Written and medically reviewed by the StethoPrep medical team.