Description |
GenCRISPR™ LbuCas13a Nuclease is an RNA-guided RNA endonuclease fromLeptotrichia buccalis. Cas13a (previously known as C2c2) belongs to the Class 2 Type VI CRISPR-Cas system and contains a single protein effector with two HEPN domains. Cas13a exhibits two distinct ribonuclease activities. Cas13a possesses sequence-specific RNA cleavage activityin vitroandin vivo. And after specific cleavage, Cas13a activates collateral cleavage activity towards adjacent non-specific ssRNA sequences. The two distinct ribonuclease activities of Cas13a enable guide-RNA processing and RNA detection. The RNA detection empowered with Cas13a-crRNA complex and quenched fluorescent ssRNA as reporter has been reported to be versatile, rapid and high-specific.
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crRNA-dependent RNA cleavage
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Source |
Recombinant Cas13a expressed byE.coli
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Species |
Leptotrichia buccalis
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Tag |
Tag-free
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Apparent Molecular Weight |
~140 kDa, on SDS-PAGE under non-reducing conditions
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Concentration |
4 mg/ml |
Storage Buffer |
10 mM Tris-HCl, 300 mM NaCl, 1.0 mM DTT, 50% glycerol, pH 8.0.
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Storage & Stability |
Store at -20 °C for up to 12 months from the date of manufacture. Avoid repeated freeze-thaw cycles.Do not store below -20 °C!
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Accession# |
C7NBY4
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Appearance |
Clear, colorless liquid
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Purity |
≥ 90% by SDS-PAGE
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Activity |
≥ 85% byin vitrodigestion of ssRNA
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Concentration |
4 mg/ml ± 10% as analyzed by A280
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Residual DNase |
Undetectable
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Residual RNase |
Undetectable
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Endotoxin Level |
< 0.2 EU/μg
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LbuCas13a has sequence-specific RNA cleavage activity, and after specific cleavage, it activates collateral cleavage activity towards adjacent non-specific ssRNA sequences.
A 50 μL reaction containing ssRNA, Cas13a/crRNA complex, Fluorescent RNA reporter, and Cas13a Reaction Buffer is incubated for 30 minutes at 37 °C. After incubation, the fluorescence intensity is measured using a Microplate Reader (Excitation wavelength: 494 nm; Emission wavelength: 518 nm).
References |
1.
Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector."Science353.6299 (2016).
2.
Gootenberg, Jonathan S., et al. "Nucleic acid detection with CRISPR-Cas13a/C2c2."Science356.6336 (2017): 438-442.
3.
Gootenberg, Jonathan S., et al. "Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6."Science360.6387 (2018): 439-444.
4.
Iwasaki, Roman S., and Robert T. Batey. "SPRINT: a Cas13a-based platform for detection of small molecules."Nucleic acids research48.17 (2020): e101-e101.
5.
East-Seletsky, Alexandra, et al. "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection."Nature538.7624 (2016): 270-273.
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For laboratory research use only. Direct human use, including taking orally and injection and clinical use are forbidden.