Frequently asked questions

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What is a PAM sequence and where is it located?

CRISPR-Cas9 mechanisms recognize DNA targets that are complementary to a short CRISPR sgRNA sequence. The part of the sgRNA sequence that is complementary to the target sequence is known as a protospacer. 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 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 sgRNA, 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 sgRNA sequence but needs to be immediately downstream of the target site in the genomic DNA.


  1. Anders C, Niewoehner O, et al. (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature, 513(7519):569–573.
  2. Sternberg SH, Redding S, et al. (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 507(7490):62–67.
  3. Esvelt KM, Mali P, et al. (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature Methods, 10(11):1116–1121.