If the target area does not contain PAM sequence, will the CRISPR/Cas9 complex bind?
No. The CRISPR-Cas9
complexes that have been characterized recognize target sequences lacking PAM sequences extremely poorly or not at all . However, if the NGG sequence found in the S. pyogenes
CRISPR system is not present in your sequence, CRISPR systems from other bacterial species that recognized alternative PAM sequences may work.
For example, the PAM sequences for S. thermophiles
are NNAGAA and NGGNG, and for N. meningiditis
, the PAM sequence is NNNNGATT [2,3]. Design your crRNA protosapcer sequence with this in mind.
- Hsu PD, Scott DA, et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology, 31(9):827–832.
- Esvelt KM, Mali P, et al. (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature Methods, 10(11):1116–1121.
- Ran FA, Hsu PD, et al. (2013) Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11):2281–2308.