Question

Which of the following protospacer adjacent motif sequences would be recognized by Cas9?

		GCG
		TGC
		ACG
		TGG
		GGT

Answer 1

CRISPR-Cas9 mechanisms recognize DNA targets that are complementary to a short CRISPR RNA (crRNA) sequence. The part of the crRNA sequence that is complementary to the target sequence is known as a spacer. In order for Cas9 to function, it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the Cas9 gene. The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG(where "N" is any nucleobase followed by two guanine ("G") nucleobases) that is found directly downstream of the target sequence in the genomic DNA, on the non-target strand.

Recognition of the PAM by the Cas9 nuclease is thought to destabilize the adjacent sequence, allowing interrogation of the sequence by the crRNA, and resulting in RNA-DNA pairing when a matching sequence is present [1,2]. Cas9 nucleases with alternative PAMs have also been characterized and successfully used for genome editing [3]. It is important to note that the PAM is not present in the crRNA sequence but needs to be immediately downstream of the target site in the genomic DNA.

TGG is the correct answer.