Leptotrichia buccalis Cas13a (LbuCas13a) 在向导 RNA 引导下识别并切割靶标 RNA或 DNA 时， 其“附属切割” 活性被激活， 可任意高效切割体系中 RNA。通过设计两端标记 荧光基团或者其他标记物的Reporter RNA，可实现 CRISPR/Cas13a 对 RNA 模板的检测和信 号放大。然后通过荧光仪或试纸观察结果。
LbuCas13a 和 LwaCas13a 都是 Cas13 家族的 RNA 靶向 CRISPR 酶。相较于LwaCas13a，LbuCas13a特异性更强。LbuCas13a常用于需要低背景、高特异性的诊断实验，例如SNV，SNP的检测。
组分表：
| 组分货号 | 组分名称 | 物理外观 | 250pmol | 1000pmol | 储存 | | L1540160A | LbuCas13a (5μM) | 液体 | 50μl | 200μl | -20℃，避免反复冻融。 | | L1540160B | Cas13a反应缓冲液 (10×) | 液体 | 1ml | 4ml | -20℃，避免反复冻融。 | | L1540160C | 稀释缓冲液 (1×) | 液体 | 1ml
| 4ml
| -20℃，避免反复冻融。 | | L1540160D | Positive Control
| 液体 | 10μl | 10μl | -20℃，避免反复冻融。 |
注：稀释缓冲液 (1×) 即蛋白稀释液。 |
需要但未提供的试剂：
1. Reporter：Cas13a切割底物。可搭配我司的Reporter进行检测，或自行设计合成Reporter.
| 货号 | 名称 |
|---|
R1540215
| ssRNA-荧光型 (Cas13a 切割底物) |
R1540216
| ssRNA-试纸型 (Cas13a 切割底物) |
2. rRNA/gRNA：与Cas13a结合，形成功能复合物，被目标序列特异性激活。推荐搭配CRISPR guide RNA转录试剂盒制备sgRNA.
LbuCas13a crRNA scaffold sequence结构序列：5’- GACCACCCCAAAAAUGAAGGGGACUAAAAC-3’
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3. 恒温扩增试剂盒：目标序列（DNA/ RNA）特异性扩增，提高目标序列与Cas13a蛋白酶结合效率。
4. T7 RNA 聚合酶: 转录RNA （货号：T292917、T1372515、T489384）
5. RNase 抑制剂（可选）：抑制RNase，防止RNA降解。（货号：R489315、R350859）
检测步骤：
1 、目标DNA/RNA特异性扩增。扩增后可使用T7 RNA 聚合酶将扩增产物转录为RNA。
注意：引物设计时需加上 T7 启动子：5’-TAATACGACTCACTATAGGG-3’
2 、根据下表配制反应体系：
| 成分名称 | 推荐用量 / 终浓度 | 优化范围 |
|---|
Cas13a反应缓冲液 (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) | | 目标 RNA/DNA** | 适量 | 0 ~ 5 μL | | H₂O | 补齐到 20 μL | - |
* 用 Diluent Buffer (1X) 稀释至合适浓度，稀释后需立即使用；
** 阳性对照组，加入 5μL Positive Control。Positive Control 已包含目标 RNA，crRNA 和 Reporter（FAM 标记）。
|
3、设置反应温度 37℃， 每 1 分钟读取一次荧光数值（FAM 荧光通道）， 反应时间 30 ~60分 钟。
注意事项：
1. Cas13a 的顺式切割：Cas13a 在 crRNA 的引导下，特异性地剪切 target RNA，且对 target RNA 的 PAM 和 PFS 序列要求并不严格。
2. Cas13a 的反式切割：当 target RNA 存在时，Cas13a/crRNA 与 target RNA 形成复合物，Cas13a 被激发反式切割活性，将反应体系中任意序列的单链 RNA 切割（如 Reporter），从而释放荧光信号。
3. 请保持实验区干净整洁，操作时需穿戴干净的手套、口罩，实验所用枪头、离心管等耗材均为 RNase-free。
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’
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.
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