Leptotrichia buccalis Cas13a (LbuCas13a) recognizes and cleaves target RNA or DNA under the guidance of guide RNA, which activates its collateral cleavage activity to efficiently cleave RNA non-specifically within the reaction system. By designing Reporter RNA labeled with fluorophores or other markers at both ends, CRISPR/Cas13a enables the detection and signal amplification of RNA templates. Results can then be read using a fluorometer or lateral flow strip.
LbuCas13a and LwaCas13a are both RNA-targeting CRISPR enzymes belonging to the Cas13 family. Compared with LwaCas13a, LbuCas13a exhibits higher specificity. LbuCas13a is widely used in diagnostic assays requiring low background and high specificity, such as the detection of SNVs and SNPs.
Components List:
| Component No. | Component Name | Appearance | 250pmol | 1000pmol | Storage | | L1540160A | LbuCas13a (5μM) | Liquid | 50μl | 200μl | -20℃. Avoid freeze/thaw cycle. | | L1540160B | Cas13a Reaction Buffer (10×) | Liquid | 1ml | 4ml | -20℃. Avoid freeze/thaw cycle. | | L1540160C | Diluent Buffer (1×) | Liquid | 1ml
| 4ml | -20℃. Avoid freeze/thaw cycle. | | L1540160D | Positive Control | Liquid | 10μl | 10μl | -20℃. Avoid freeze/thaw cycle. |
Note: Diluent Buffer (1×) refers to the protein dilution buffer. |
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/gRNA: Binds to Cas13a to form a functional complex, which is specifically activated by target sequences. sgRNA preparation is recommended using the CRISPR guide RNA Transcription Kit.
LbuCas13a crRNA scaffold sequence: 5’- GACCACCCCAAAAAUGAAGGGGACUAAAAC-3’
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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 DNA/RNA. After amplification, T7 RNA Polymerase can be used to transcribe the amplicons into RNA.
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× | - | | Reporter (fluorescence) | 120 nM | 40 ~ 2000 nM | | crRNA | 500 nM | 100 ~ 1000 nM | | LbuCas13a * | 125 nM (=0.125 μM) | 100 ~ 500 nM (=0.1~0.3 μM) | | Target RNA/DNA** | Appropriate volume | 0 ~ 5 μL | | H₂O | Bring total volume to 20 μL | - |
* Dilute to the appropriate concentration with 1× Diluent Buffer, and use immediately after dilution.
** For the positive control group, add 5 μL Positive Control. The Positive Control contains target RNA, crRNA and FAM-labeled Reporter. |
3. Incubate the reaction at 37°C, record fluorescence readings every 1 minute (FAM channel), total reaction time: 30–60 min.
Precautions
1. Cis-cleavage of Cas13a: Under the guidance of crRNA, Cas13a specifically cleaves target RNA, and has loose requirements for the PAM and PFS sequences of target RNA.
2. Trans-cleavage of Cas13a: In the presence of target RNA, Cas13a/crRNA forms a complex with target RNA. Cas13a is activated to exert trans-cleavage activity, which cleaves single-stranded RNA of arbitrary sequences in the reaction system (e.g. Reporter), thereby releasing fluorescent signals.
3. Keep the experimental area clean. Wear clean gloves and masks during operation. Tips, centrifuge tubes and other consumables used in the experiment must be RNase-free.
