AapCas12b 是 Type V 亚型的 Cas 核酸酶，在 37~60℃都具有良好活性。在 crRNA 和 tracrRNA（或两者连接形成的 sgRNA）引导 下，对双链和单链 DNA 靶标均能产生顺式切割活性 (cis -cleavage activity)。对于双链 DNA 靶标，AapCas12b 识别靶标 DNA 中 PAM 序列 (5'-TTN) 下游与 crRNA（或 sgRNA）spacer 序列互补的位置， 特异性切割靶标双链 DNA 并生成粘性末端；对于单链 DNA 靶标，其特异性切割不依赖 PAM 序列。 AapCas12b、靶标 DNA、sgRNA 形成三元复合物后，还会产生反式切割活性 (trans -cleavage activity)，即非特异性切割任意序列 的单链 DNA。AapCas12b 在常见的等温扩增反应缓冲液中也具有良好活性。
组分表
| A1522667 | 组件 | 物理外观 | 100pmol | 1000pmol | 储存 | | A1522667A | AapCas12b (10 μM) | Liquid | 10μl | 100μl | -20℃. Avoid freeze/thaw cycle. | | A1522667B | 10× Cas12b Buffer | Liquid | 1ml | 1ml | -20℃. Avoid freeze/thaw cycle. |
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质量控制
蛋白纯度
使用 SDS-PAGE 凝胶电泳检测，蛋白纯度不低于 95%。
核酸内切酶残留检测
将 10 pmol AapCas12b 与 200 ng 超螺旋质粒 DNA 在 37℃温 育 4 h，使用琼脂糖凝胶电泳检测，少于 10% 的质粒 DNA 转变成缺 刻或线性状态。
非特异性核酸酶残留检测
将 10 pmol AapCas12b 与 15 ng 双链 DNA 片段在 37℃温育 16 h，使用琼脂糖凝胶电泳检测双链 DNA 底物无变化。
RNase 残留检测
将 10 pmol AapCas12b 与 500 ng 总 RNA 在 37℃温育 1 h，使 用琼脂糖凝胶电泳检测超过 90% 的 RNA 仍保持完整。
宿主 DNA 残留检测
使用大肠杆菌 16S rDNA 特异性引物探针组，采用荧光定量 PCR 法检测 10 pmol AapCas12b，大肠杆菌宿主基因组 DNA 残留低于 1 copy。
失活条件
85℃温浴 5 min。
使用方法
1. 顺式切割实验
① 在冰上配制如下反应体系：
| 试剂 | 使用量 | 终浓度 |
|---|
| 10× Cas12b Buffer | 2 μl | 1× | | AapCas12b (10 μM)ᵃ | 0.5 μl | 250 nM | | sgRNA (10 μM)ᵃ | 0.5 μl | 250 nM | | Target DNA (1 μM)ᵃ | 0.5 μl | 25 nM | | Nuclease-free water | To 20 μl | — |
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a. 如果用凝胶电泳检测顺式切割产物，建议使用双链 DNA 作为靶标，长度在 300 bp~3 kb 之间。建议保持 AapCas12b : sgRNA : Target DNA 的摩尔比为 10:10:1，尽量确保靶标 DNA 被完全切割。
② 60℃反应 30 min~1 h 后，85℃孵育 5 min 灭活；
③ 使用琼脂糖凝胶电泳检测产物。为避免 AapCas12b 与 DNA 结合导致电泳异常，请确保进行热失活；若仍有电泳异常情况，可使用蛋白酶 K 对产物处理之后再进行电泳分析。
2. 反式切割实验
① 在冰上配制如下反应体系：
| 试剂 | 使用量 | 终浓度 |
|---|
| 10× Cas12b Buffer | 2 μl | 1× | | AapCas12b (10 μM)ᵃ | 0.05~0.5 μl | 25~250 nM | | sgRNA (10 μM)ᵃ | 0.05~0.5 μl | 25~250 nM | | Target DNA (1 μM)ᵇ | 0.5~5 μl | 25~250 nM | | ssDNA Probe (10 μM)ᵃ | 0.05~0.5 μl | 25~250 nM | | Nuclease-free water | To 20 μl | — |
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a. 反式剪切实验中各组分的用量可以根据实验目的调整，用量较少时可先稀释成预混液。
b. 反式切割实验中的靶标 DNA 可以是单链 DNA 或带 PAM 序列的双链 DNA。
② 置于荧光定量 PCR 仪中，根据单链 DNA 探针所带荧光基团选择对应通道，60℃恒温反应，每 30 s 为 1 个循环。循环数可设置为 30~60 个。
AapCas12b is a Cas nuclease of the Type V subtype, with robust activity between 37°C and 60°C. Guided by crRNA and tracrRNA (or sgRNA formed by the two), it exhibits cis-cleavage activity against both double-stranded and single-stranded DNA targets. For double-stranded DNA targets, AapCas12b recognizes the position complementary to the spacer sequence of crRNA (or sgRNA) downstream of the PAM sequence (5'-TTN) in the target DNA, and specifically cleaves the target double-stranded DNA to generate sticky ends. For single-stranded DNA targets, its specific cleavage is independent of the PAM sequence. After forming a ternary complex with the target DNA and sgRNA, AapCas12b also exhibits trans-cleavage activity, i.e., non-specific cleavage of single-stranded DNA of any sequence. AapCas12b also shows good activity in common isothermal amplification reaction buffers.
Components List
| A1522667 | Components | Appearance | 100pmol | 1000pmol | Storage | | A1522667A | AapCas12b (10 μM) | Liquid | 10μl | 100μl | -20℃. Avoid freeze/thaw cycle. | | A1522667B | 10× Cas12b Buffer | Liquid | 1ml | 1ml | -20℃. Avoid freeze/thaw cycle. |
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Quality Control
- Protein PurityDetermined by SDS-PAGE, the protein purity is ≥95%.
- Endonuclease Residue Detection10 pmol of AapCas12b is incubated with 200 ng of supercoiled plasmid DNA at 37℃ for 4 h. As determined by agarose gel electrophoresis, less than 10% of the plasmid DNA is converted to nicked or linear form.
- Non-specific Nuclease Residue Detection10 pmol of AapCas12b is incubated with 15 ng of double-stranded DNA fragments at 37℃ for 16 h. No change in the double-stranded DNA substrate is observed by agarose gel electrophoresis.
- RNase Residue Detection10 pmol of AapCas12b is incubated with 500 ng of total RNA at 37℃ for 1 h. More than 90% of the RNA remains intact as determined by agarose gel electrophoresis.
- Host DNA Residue DetectionUsing E. coli 16S rDNA-specific primer-probe set, the residual host genomic DNA in 10 pmol of AapCas12b is detected by qPCR and is less than 1 copy.
Inactivation Condition
Incubate at 85℃ for 5 min.
Protocol
1. Cis Cleavage Assay
① Prepare the following reaction system on ice:
| Reagent | Volume | Final Concentration |
|---|
| 10× Cas12b Buffer | 2 μl | 1× | | AapCas12b (10 μM)ᵃ | 0.5 μl | 250 nM | | sgRNA (10 μM)ᵃ | 0.5 μl | 250 nM | | Target DNA (1 μM)ᵃ | 0.5 μl | 25 nM | | Nuclease-free water | To 20 μl | — |
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ᵃ. If the cleavage products are detected by gel electrophoresis, it is recommended to use double-stranded DNA as the target, with a length between 300 bp and 3 kb. It is recommended that the molar ratio of AapCas12b : sgRNA : Target DNA is 10:10:1 to ensure complete cleavage of the target DNA as much as possible.
② Incubate at 60℃ for 30 min~1 h, then inactivate at 85℃ for 5 min.
③ Analyze the products by agarose gel electrophoresis. To avoid abnormal electrophoresis caused by AapCas12b binding to DNA, ensure heat inactivation is performed. If electrophoresis abnormalities still occur, treat the products with Proteinase K before electrophoresis analysis.
2. Trans Cleavage Assay
① Prepare the following reaction system on ice:
| Reagent | Volume | Final Concentration |
|---|
| 10× Cas12b Buffer | 2 μl | 1× | | AapCas12b (10 μM)ᵃ | 0.05~0.5 μl | 25~250 nM | | sgRNA (10 μM)ᵃ | 0.05~0.5 μl | 25~250 nM | | Target DNA (1 μM)ᵇ | 0.5~5 μl | 25~250 nM | | ssDNA Probe (10 μM)ᵃ | 0.05~0.5 μl | 25~250 nM | | Nuclease-free water | To 20 μl | — |
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ᵃ. The amount of each component in the trans-cleavage assay can be adjusted according to the experimental purpose. When using small amounts, it is recommended to prepare a master mix by dilution first.
ᵇ. The target DNA in the trans-cleavage assay can be single-stranded DNA or double-stranded DNA with a PAM sequence.
② Place the reaction in a qPCR instrument, select the corresponding channel according to the fluorophore of the ssDNA probe, and perform a constant temperature reaction at 60℃, with each cycle set to 30 s. The number of cycles can be set to 30~60.
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