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Applications: Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.
Assay Conditions: Varying amounts of PsmCas13b activity was measured using a CRISPR-based fluorescent reporter assay for optimal results. Target RNA cutting and collateral RNase activity was activated using RNA-guided RNA Binding to Cas13a. Emission of fluorescent signal is due to the degradation of the reporter substrate upon cleavage.Active Cas13 was thawed on ice while 1X Reaction Buffer containing 20 mM HEPES, pH 7.0, 50 mM KCl, 5 mM MgCl2, and 0.1 mg/ml BSA, guide RNA (target-specific spacer sequence), target RNA activator (complementary sequence to crRNA) were equilibrated to room temperature. Next three working solutions of Active Cas13 (4X final concentration) guide RNA (4X final concentration) and activator/reporter mix containing RNA activator and reporter substrate (2X final concentration), were prepared using 1X Reaction Buffer. 10 µl of 4X active Cas12 and 10 µl of 4x guide RNA were then preincubated in half the area of a solid black 96-well plate for 5 minutes at room temperature. After preincubation, 20 µl of 2X activator/reporter mix was added to plate and placed on shaking incubator for 1 min. The plate was then sealed and incubated at 37°C for 10-30 minutes. Plate was then equilibrated to room temperature, plate sealer removed and fluorescence read on a microplate reader. Negative control was measured by replacing enzyme working solution with equal volume of assay buffer.
Description: Recombinant Prevotella sp MA2016 PsmCas13b (type VI-B CRISPR-associated RNA-guided ribonuclease Cas13b), full length. This construct contains an N-terminal His-tag. Psm Cas13b is a CRISPR-Cas effector that belongs to the class 2 subtype VI-B and effectively targets and silences disting mammalian ssRNA virus.
Format: Aqueous buffer solution.
Formulation: 50 mM Tris, pH 7.5, 600 mM NaCl, 2 mM DTT, and 10% glycerol
Genbank: WP_028912271
Purity: ≥90%
Storage Stability: At least 6 months at -80°C.
Tags: N-terminal His-tag
Warnings: Avoid multiple freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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