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What is a PAM sequence and where is it located?

CRISPR-Cas 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 protospacer. For a CRISPR-Cas enzyme to function it also requires a specific protospacer adjacent motif (PAM) that varies depending on the bacterial species of the enzyme.

The most commonly used Cas9 nuclease, derived from S. pyogenes, recognizes a PAM sequence of NGG that is directly downstream of the target sequence on the non-target strand of genomic DNA.

Comparison of RNP: CRISPR-Cas9 vs. CRISPR-Cpf1

 

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].

Other Cas nucleases with alternative PAMs have also been characterized and successfully used for genome editing [3], including the TTTV PAM site found in the Cpf1 system (Alt-R® CRISPR-Cpf1 System). 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.

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. Nat Methods, 10(11):1116–1121.
  4. Zetsche B, Gootenberg JS, et al. (2015) Cpf1 is a Single RNA-guided endonuclease of a Class 2 CRISPR-Cas system. Cell, 163(3):759–771.
Tags:
  • Cas9
  • Cpf1
  • protospacer
  • PAM
  • CRISPR
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