Cas13a, Recombinant, L. buccalis

Cas13a is an RNA-guided endonuclease that belongs to the class 2 type VI CRISPR-Cas system. When activated, Cas13a induces collateral cleavage of nearby non-targeted RNAs in a nonspecific manner.||Full length recombinant protein corresponding to L. buccalis Cas13a, expressed in E. coli. ||Molecular Weight: |~130kD||Assay Conditions:|Varying amounts of LbuCas13a 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. 10ul of 4X active Cas12 and 10ul 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, 20ul 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.||Applications:|Useful for the study of enzyme kinetics, screening inhibitors, and selectivity profiling.||Storage and Stability:|Aliquot to avoid repeated freezing and thawing and store at -70°C. Aliquots are stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.