Frequently asked questions

Our Scientific Applications Support team has assembled a list of frequently asked questions to help you find answers quickly. Filter using one or more categories to focus on specific topics, or use the search bar to perform a text search.

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If the target area does not contain a protospacer adjacent motif (PAM) sequence, will the CRISPR-Cas9 complex bind?

No, CRISPR-Cas9 complexes do not recognize, or recognize extremely poorly, targets lacking PAM sequences [1].

The PAM sequence recognized by the S. pyogenes CRISPR-Cas9 system is NGG. If this sequence is not present in your target, you may be able to use other CRISPR systems (from other bacterial species) that recognize different PAM sequences. The following table lists examples of PAM sequences:

Bacterial species PAM sequence Reference
S. pyogenes (Cas9)* NGG [1]
Acidaminococcus sp BV3L6 (Cpf1)* TTTV [2]
Streptococcus thermophilus NNAGAA
NGGNG
[3,4]
Neisseria meningitidis NNNNGATT [3,4]

* In addition to the Alt-R® CRISPR-Cas9 System, we offer the Alt-R CRISPR-Cpf1 System (crRNA, nuclease, and electroporation enhancer). Note that each CRISPR nuclease has unique requirements for crRNA and electroporation enhancer design. You will not be able to use Cas9 and Cpf1 reagents in the same reaction.

References

  1. Hsu PD, Scott DA, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 31(9):827–832.
  2. 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.
  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. Ran FA, Hsu PD, et al. (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 8(11):2281–2308.