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 . While Jiang et al. have shown that S. pyogenes Cas9 can also recognize NAG, it does so less efficiently .
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.
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 .
Keep these options in mind when selecting crRNA protospacer sequences.
Learn more about the Alt-R CRISPR Systems here.