TriAltus' highly pure Cas12b protein is used for genome editing with CRISPR technology and CRISPR-mediated in vitro applications. Recombinant Alicyclobacillus acidiphilus Cas12b (AapCas12b) protein is expressed in E. coli with two C-terminal nuclear localization signals (NLS) and the CL7 tag, which is removed with PreScission protease during the purification process. AapCas12b protein combines with guide RNA (gRNA) to form a stable ribonucleoprotein (RNP) complex that enables the inactive nuclease to be guided to the specific double stranded DNA (dsDNA) site. Once guided to the specific cleavage site, AapCas12b becomes activated to specifically and precisely cleave the dsDNA and to indiscriminately trans-cleave single stranded DNA (ssDNA) nearby.
PURITY | ≥90%
ENZYME SOURCE | Alicyclobacillus acidiphilus Cas12b is expressed in E. coli with two C-terminal nuclear localization signals (NLS) and is purified with our CLīM technology.
STORAGE/SHIPPING CONCENTRATION | ~ 5 mg/mL
STORAGE BUFFER | 10 mM Tris-Cl pH 8.0, 250 mM NaCl, 50% glycerol
RECOMMENDED STORAGE CONDITIONS | -80°C
EXPIRATION | 6 months from receipt when stored as directed. Avoid repeated freeze/thaw cycles.
Figure 1. CLīM-purified AapCas12b’s trans-cleavage nuclease activity compared to commercially obtained (“C.O.”) AapCas12b. Upon hybridization of the Cas12b/sgRNA RNP to the target, dsDNA activator, Cas12b becomes active to cleave the activator as well as to trans-cleave nearby nonspecific ssDNA. Activated Cas12b cleaves dual-labeled fluorophore and quencher (FQ)-ssDNA Reporter, which releases the fluorophore to emit a fluorescent signal. Raw RFU values from each reaction were corrected by subtracting the background fluorescence value of the appropriate “no activator” control. The average of the corrected RFU values and SEM are reported. Each condition was performed in technical triplicates.
AapCas12b obtained from a commercial source or CLīM-purified AapCas12b was combined with annealed sgRNA for 10 minutes at room temperature to create functional AapCas12b RNP complexes. Reaction buffer was used in the place of protein for the “No Cas12b” control. A mixture of target dsDNA activator and a nonspecific FQ-ssDNA reporter was added to the designated RNP reactions. The reaction proceeded for 30 minutes at 37°C, equilibrated to room temperature for 10 minutes, and the fluorescence was measured. The reaction systems comprised the following: 0 or 40nM AapCas12b, 44nM sgRNA, 0 or 1 nM Activator, 100 nM FQ-Reporter and 1x NEBuffer r2.1 (New England Biolabs).
20 µg: 40-1212
100 µg: 40-1220
500 µg: 40-1225