Cas9 Nuclease, S. pyogenes, is an RNA-guided endonuclease that catalyzes site-specific cleavage of double stranded DNA. The location of the break is within the target sequence 3 bases from the NGG PAM (Protospacer Adjacent Motif) (1). The PAM sequence, NGG, must follow the targeted region on the opposite strand of the DNA with respect to the region complementary sgRNA sequence.
Cas9 in vivo: Bacterial Adaptive Immunity
CRISPR loci in the bacterial genome are transcribed and processed into crRNA. Cas9 endonuclease complexed with a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.)
Cas9 nuclease, S. pyogenes, complexed with an sgRNA
Cleavage occurs three nucleotides upstream of the PAM sequence (shown in red). sgRNAs are complimentary to the strand of DNA opposite of the PAM.
In vitro DNA cleavage using Cas9 Nuclease, S. pyogenes
Reactions were set up according to recommended conditions, and cleavage products were resolved on a 1% agarose TBE gel. Input DNA is PvuII-linearized pBR322 plasmid DNA.
Product Source
A His-tagged engineered variant of E. coli that carries the cloned Cas9 gene from Streptococcus pyogenes.
This product is related to the following categories:
If planning to use higher concentration Cas9 Nuclease, S. pyogenes (M0386T and M0386M) for in vitro digestion of DNA, the enzyme should be diluted to 1 µM using Diluent B (NEB #B8002S) prior to the reaction assembly.
References
Jinek M. et al. (2012). Science. 816-821 doi: 10.1126/Science.1225829. Epub 2012 Jun 28.. PubMedID: 22745249
Haussmann IU, Ustaoglu P, Brauer U, Hemani Y, Dix TC, Soller M. (2018) Plasmid-based gap-repair recombineered transgenes reveal a central role for introns in mutually exclusive alternative splicing in Down Syndrome Cell Adhesion Molecule exon 4 Nucleic Acids Res; Dec 12, PubMedID: 30541104, DOI: 10.1093/nar/gky1254
Lee, NC., Larionov, V., Kouprina, N. (2015) Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast Nucleic Acids Res; 43(8), e55. PubMedID: 25690893
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