When Cas13a recognizes and cleaves target RNA under the guidance of guide RNA, its collateral cleavage activity is activated, enabling efficient non-specific cleavage of RNA present in the reaction system. By designing Reporter RNA labeled with fluorophores or other markers at both ends, CRISPR/Cas13a can be applied for the detection and signal amplification of RNA templates. Results can then be read using a fluorometer or lateral flow strip.
LwaCas13a (C2c2) is a member of the Cas13a protein family, derived from Leptotrichia wadei. Upon binding to crRNA, it specifically recognizes RNA sequences and performs trans-cleavage of RNA. Compared with LbuCas13a, LwaCas13a exhibits high activity and faster cleavage kinetics in certain diagnostic assays.
Components List:
| Component No. | Component Name | Appearance | 500pmol | 2000pmol | Storage | | L1540159A | LwaCas13a (10μM) | Liquid | 50μl | 200μl | -20℃. Avoid freeze/thaw cycle. | | L1540159B | Cas13a Reaction Buffer (10×) | Liquid | 1ml | 4ml | -20℃. Avoid freeze/thaw cycle. | | L1540159C | Diluent Buffer (1×) | Liquid | 500μl | 4ml | -20℃. Avoid freeze/thaw cycle. |
Note:
1. LwaCas13a is supplied in storage buffer containing 5% glycerol.
2. Diluent Buffer (1×) is for LwaCas13a protein dilution, containing 5% glycerol.
3. Glycerol-free formulation is available upon custom request for lyophilization applications. |
Other materials required:
1. Reporter: Cas13a cleavage substrate. Detection can be performed using our Reporter products, or you may design and synthesize your own Reporter.
Cat. No.
| Item |
|---|
| R1540215 | FAM-BHQ1 labeled ssRNA (Cas13a Fluorescent Reporter)
|
| R1540216 | FAM-Biotin labeled ssRNA (Cas13a Lateral Flow Reporter) |
2. crRNA/sgRNA: Binds to Cas13a to form a functional complex, which is specifically activated by target sequences. (LwaCas13a crRNA scaffold sequence: 5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3’) sgRNA preparation is recommended using the CRISPR guide RNA Transcription Kit.
3. Isothermal amplification kit: Specifically amplifies target sequences (DNA/RNA) to improve the binding efficiency between target sequences and LwaCas13a protein.
4. T7 RNA Polymerase: For RNA transcription (Cat. No.: T292917, T1372515, T489384)
5. RNase Inhibitor (optional): Inhibits RNase to prevent RNA degradation. (Cat. No.: R489315, R350859)
Assay Protocol:
1. Specific amplification of target sequence (DNA/RNA). Amplification methods including PCR, RPA, RAA, LAMP, RCA are applicable.
Note: The T7 promoter sequence (5’-TAATACGACTCACTATAGGG-3’) must be incorporated into the primer design.
2. Prepare the reaction system according to the table below:
| Component | Recommended Amount / Final Concentration | Optimization Range |
|---|
| Cas13a Reaction Buffer (10×) | 1× | - | | 10 mM NTPs | 1 mM each | - | | Reporter (fluorescence) | 120 nM | 40 ~ 160 nM | | crRNA | 20 nM | 20 ~ 80 nM | | LwaCas13a | 100 nM (=0.1 μM) | 40 ~ 160 nM (=0.04~0.16 μM) | | RNase Inhibitor | 40 U total | - | | T7 RNA Polymerase | 30 U total | 5 ~ 50 U | | Amplicons | Appropriate volume | 0–5 μL | | H₂O | Bring total volume to 20 μL | - |
*Example: Mix 1 μL of 10 μM LwaCas13a with 9 μL dilution buffer, then add 2 μL of the diluted protein into the 20 μL reaction system, to reach a final Cas protein concentration of 100 nM.
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3. Incubate the reaction at 37°C, record fluorescence readings every 1 minute for a total reaction time of 30–60 min.
Precautions
1. For research use only. Please read this manual carefully before use.
2. Operate in strict accordance with the management specifications for gene amplification laboratories to prevent amplicon contamination.
3. Waste generated during the assay shall be disposed of in accordance with relevant regulations after the experiment.
