Intact Genomics Cas12a Nuclease is the purified recombinant Francisella tularensis Cas12a enzyme for in vitro gene editing.
$154.00 – $525.00
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids (1). The CRISPR system consists of a short non-coding guide RNA (sgRNA) made up of a target complementary CRISPR RNA (crRNA) and an auxiliary transactivating crRNA (tracrRNA). The sgRNA guides the Cas12a (Cpf1) endonuclease to a specific genomic locus via base pairing between the crRNA sequence and the target sequence, and cleaves the DNA to create a “sticky” double-strand break with a four base pair overhang, which is different from the Cas9 generated blunt end break. location of the break is within the target sequence 18 bases from the TTTV (V is A, C or G) PAM (Protospacer Adjacent Motif) (2). The PAM sequence must precede the targeted region on the opposite strand of the DNA with respect to the region complementary sgRNA sequence (Fig.1).
Intact Genomics (ig®)Cas12a Nuclease is the purified recombinant Francisella tularensis Cas12a enzyme for in vitro editing. This enzyme is designed to perform CRISPR/Cas12a-mediated genome editing (1-3). The physical purity of this enzyme is ≥65% as assessed by SDS-PAGE with Coomassie® blue staining and densitometry.
E.coli BL21 (DE3) strain expressing a Cas12a (Cpf1) gene from Francisella tularensis with an N-terminal 6xHis tag.
- Cas12a Nuclease
- 10x Cas12a Nuclease Reaction Buffer
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 °C
1x Cas9 Reaction Buffer
20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, pH 6.5 @ 25 °C
Quality Control Assays
Cas9 nuclease is free from detectable RNase, Endonuclease (nicking) and non-specific DNase activities.3273 3276
80 µg (1600ng/µl), 400 µg (1600ng/µl)
Cas12a Nuclease functional testing was done by in vitro DNA cleavage assay with the following protocol which gives more than 50% digestion of the substrate DNA as determined by agarose gel electrophoresis.
- Set up 30 µl reaction in a microcentrifuge tube on ice with the following combinations.
- Gently mix the reaction mixture and centrifuge briefly.
- Incubate at 37 °C for 60 min.
- Add 1 µl RNase (4 mg/ml)
- Incubate at 37 °C for 20 min.
- Run 0.7 to1% agarose TBE gel.