Cas13a 在向导 RNA 引导下识别并切割靶标 RNA 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。
LwaCas13a (C2c2) 是Cas13a家族蛋白成员，来源于Leptotrichia wadei。与crRNA结合，特异性识别RNA序列，反式切割RNA。相较于LbuCas13a, LwaCas13a 活性很强，在一些诊断实验中切割速度更快。
组分表：
| 组分货号 | 组分名称 | 物理外观 | 500pmol | 2000pmol | 储存 | | L1540159A | LwaCas13a (10μM) | 液体 | 50μl | 200μl | -20℃，避免反复冻融。 | | L1540159B | Cas13a反应缓冲液 (10×) | 液体 | 1ml | 4ml | -20℃，避免反复冻融。 | | L1540159C | 稀释缓冲液 (1×) | 液体 | 500μl | 2ml | -20℃，避免反复冻融。 |
注：
1. LwaCas13a采用含 5% 甘油的储存液保存。
2. 稀释缓冲液 (1×) 为 Cas13a 蛋白稀释液，含 5% 甘油。
3. 如用于冻干应用，可提供无甘油版本定制服务。 |
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. crRNA/sgRNA：与Cas13a结合，形成功能复合物，被目标序列特异性激活。
(LwaCas13a crRNA scaffold sequence结构序列： 5’-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAAC-3’) 推荐搭配CRISPR guide RNA转录试剂盒制备sgRNA。
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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 、目标序列（DNA/RNA）特异性扩增。可以采用PCR，RPA，RAA，LAMP，RCA等扩增技术。 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× | - | | 10 mM NTPs | 1 mM each | - | | Reporter (fluorescence) | 120 nM | 40 ~ 160 nM | | crRNA | 20 nM | 20 ~ 80 nM | | LwaCas13a | 100 nM*(=0.1uM) | 40 ~ 160 nM (=0.04~0.16μM) | RNase 抑制剂
| 40 U total | - | T7 RNA 聚合酶
| 30 U total | 5 ~ 50 U | | 扩增产物 | 适量 | 0-5μl | | H₂O | 补齐到 20 μL | - |
* 例如10μM LwaCas13a 1μL + 稀释缓冲液 9μL，取2μL 加入20μL 的反应体系，此时Cas蛋 白的终浓度为100nM. |
3、设置反应温度 37℃， 每 1 分钟读取一次荧光数值， 反应时间 30~60分钟。
注意事项：
1. 本产品仅供科研使用， 使用前请仔细阅读本说明书。
2. 请严格按照基因扩增实验室的管理规范进行实验操作， 注意防止扩增产物污染。
3. 实验结束后， 检测过程中所产生的废弃物应按照相关规范进行处理。
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.
|
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.
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