No Cas9 PAM NGG sequence in your target region for genome editing?

A restricted or AT-rich target region for genome editing experiments may lack a PAM NGG motif recognized by S. pyogenes Cas9. The Acidaminococcus sp. BV3LC Cpf1 enzyme uses a PAM site of TTTV (i.e., TTTA, TTTC, TTTG), making it especially useful for targeting AT-rich regions. Read about this and other alternatives for targeting sequences with few or no NGG sites.

Mar 24, 2016

Revised/updated Sep 28, 2017

Occasionally, researchers performing genome editing are confronted with a narrow target region that lacks the recommended Cas9 NGG protospacer adjacent motif (PAM). S. pyogenes Cas9 endonuclease, the enzyme most commonly used for these experiments, does not effectively recognize target sequences lacking an NGG [1]. While Jiang et al. have shown that S. pyogenes Cas9 can also recognize NAG, it does so less efficiently [2].

The Acidaminococcus sp. BV3LC Cpf1 enzyme uses a PAM site of TTTV (i.e., TTTA, TTTC, TTTG). It is therefore, especially useful for targeting AT-rich regions. Unlike S. pyogenes Cas9, which cleaves most NGG PAM sites to some degree, some of the tested TTTV sites show no cleavage by A.s. Cpf1 nuclease (Figure 1). We, therefore, strongly recommend using positive control crRNAs to establish that your cells can be edited by Cpf1. In addition, we recommend testing 3 or more crRNAs per target gene.

D-GE16PT-cpf1 PAM site-F1

Figure 1. Maximize successful CRISPR-Cpf1 genome editing by using TTTA, TTTC, or TTTG as the PAM site. HEK-293 cells were transfected with ribonucleoprotein (RNP: Alt-R A.s. Cpf1 Nuclease 2 NLS complexed with Alt-R CRISPR-Cpf1 crRNA; IDT) as instructed in the Alt-R CRISPR-Cpf1 User Guide—RNP electroporation, Amaxa Nucleofector system. TTTN sites from 6 genes were used to design 232 Alt-R CRISPR-Cpf1 crRNAs. Editing efficiency was determined 48 hr after electroporation using the Alt-R Genome Editing Detection Kit, which provides the major components required for T7EI endonuclease assays. PAM = protospacer adjacent motif (Cpf1 PAM sequence is TTTV, where V = A, C, or G); N = any base.

The A.s. Cpf1 nuclease is provided by IDT as part of the Alt-R CRISPR-Cpf1 System, for use in electroporation experiments. We recommend using the Alt-R A.s. Cpf1 Nuclease 2NLS combined with the Alt-R CRISPR-Cpf1 crRNA to generate a ribonucleoprotein (RNP) editing complex. The Alt-R Cpf1 Electroporation Enhancer is critical for optimal delivery by electroporation and is recommended for all experiments using this transfection method. For further guidance on electroporation and delivery of the RNP into your cell lines, see the Resources section of the Alt-R CRISPR-Cpf1 System webpage.

In addition, Cas9 from S. thermophiles recognizes the PAM sequences NNAGAA and NGGNG [3, 4], while Cas9 from N. meningitidis recognizes NNNNGATT and NNNNGCTT, albeit the latter enzyme requires a 24 nt protospacer [5].

Keep these options in mind when selecting crRNA protospacer sequences.

Learn more about the Alt-R CRISPR Systems here.


  1. Hsu PD, Scott DA, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 31(9):827–832.
  2. Jiang et al. (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol, 31:233–239.
  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.
  5. Hou Z, et al. (2013) Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA 110:15644–15649.