The guide molecule contains a sequence (protospacer) that is complementary to the DNA segment targeted. Immediately adjacent to this protospacer is another short critical sequence known as the protospacer adjacent motif (PAM). The PAM is distinct from the complementary target sequence component of sgRNA and is downstream of the target site found in the genomic DNA.1
When constructing sgRNA guide molecules, the PAM sequence must be positioned on the 3’ end of the DNA target sequence after the crRNA and tracrRNA sequences. The Cas9
nuclease mechanism is initiated when it recognizes the PAM and proceeds to destabilize the adjacent sequences to allow DNA pairing with the complementary sequence found on the sgRNA.
1. C. Anders, et al., “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature