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.

pam-site-callout

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.

References:

  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.