Cas14a是一种内切核酸酶，其在tracrRNA:crRNA（或sgRNA）的引导下，特异结合并切割靶标ssDNA，且无需PAM位点。与Cas12a类似，Cas14a也可以结合靶标核酸并激活其 ssDNA反式切割活性，从而被应用于靶标核酸的分子检测；与Cas12a不同的是，Cas14a只能结合ssDNA靶标，因此经过扩增富集的靶标核酸需使用T7核酸外切酶进行处理，且其中一条扩增引物需要使用磷硫酰化修饰以确保T7核酸外切酶仅切割其中一条链，从而留下ssDNA靶标链以用于Cas14a介导的分子检测。
Cas14可实现高保真SNP基因分型。由于对DNA分子的识别不依赖PAM位点，Cas14对识别序列的准确性要求极其严格，单碱基的错配就会严重抑制其切割活力。
组分表：
| 组分货号 | 组分名称 | 物理外观 | 100pmol | 1000pmol | 储存 | | C1540161A | Cas14a (10μM) | 液体 | 10μl | 100μl | -20℃，避免反复冻融。 | | C1540161B | Cas14a稀释缓冲液 (1×) | 液体 | 100μl | 1ml | -20℃，避免反复冻融。 | | C1540161C | Cas14a反应缓冲液 (10×) | 液体 | 200μl | 2×1ml
| -20℃，避免反复冻融。 | | C1540161D | Positive Control | 液体 | 10μl | 10μl | -20℃，避免反复冻融。 |
* Cas14a稀释缓冲液 (1×) 即蛋白稀释液。 |
Cas14a sgRNA:
Cas14a sgRNA由tracrRNA, crRNA和Spacer 构成。sgRNA与Cas14a结合，形成功能复合物，被目标序列特异性激活。
Cas14a sgRNA scaffold sequence结构序列: 5’-3’:
CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAG UGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUC GAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAA
UGAAGGAAUGCAAC
推荐搭配CRISPR guide RNA转录试剂盒制备sgRNA。
.png)
检测步骤：
1. 根据下表配制Cas14a切割反应体系：（建议冰上操作）
| 组分 | 用量 | 终浓度 |
|---|
Cas14a反应缓冲液 (10×)
| 2 μl | 1X | | Cas14a (10μM)* | 0.5~2 μl | 250~1000 nM | | 10 μM sgRNA | 0.5~2 μl | 250~1000 nM | | 10 μM Target DNA** | X μl | - | | 10 μM ssDNA Reporter (for Cas14a)*** | 0.5~2 μl | 250~1000 nM | | Nuclease-free water | Up to 20 μl | - |
* 可用 Cas14a稀释缓冲液 (1×) 稀释，稀释后需立即使用。
**Target DNA 可为 ssDNA 或带 PAM 序列的 dsDNA，Positive Control 包含 sgRNA、Reporter 和 ssDNA，20μL 反应体系加入 5μL。（crRNA、Target DNA 及 ssDNA Reporter 可用 Nuclease-free Water 稀释。对于极低浓度的 Target DNA，建议用 0.1% Tween 20 稀释并使用低吸附的离心管、吸头等耗材。）
*** 推荐阿拉丁 Reporter for Cas14a。(Cat#: R1540217 ssDNA-荧光型 (Cas14a 切割底物)) 它带有FAM荧光标签。 |
2. 设置 Q-PCR 仪温度为 37℃，每分钟采集一次荧光信号。
.png)
注意事项
1. 如果使用 Q-PCR 仪，请关闭热盖功能或将热盖温度设置为 42℃。
2. Cas14a 的顺式剪切 (cis-cleavage)：Cas14a 在 sgRNA 的引导下，特异性地剪切 target DNA。dsDNA 靶标需带有 PAM 位点，而 ssDNA 靶标不依赖 PAM 位点。
3. Cas14a 的反式剪切 (trans-cleavage)：当 target DNA 存在时，Cas14a/sgRNA 与 target DNA 形成复合物，Cas14a 被激发反式剪切活性，将反应体系中任意序列的单链 DNA 切割。
4. 当 Cas14a 结合靶标 ssDNA 时，需使用 T7 核酸外切酶对扩增得到的靶标 dsDNA 进行消化，得到靶标 ssDNA，用于 Cas14a 介导的分子检测。（注意：其中一条扩增引物需要采用磷硫酰化修饰以确保 T7 核酸外切酶仅切割另外一条链，从而留下 ssDNA 靶标链。）
5. 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。
Cas14a is an endonuclease. Guided by tracrRNA:crRNA (or sgRNA), it specifically binds to and cleaves target ssDNA without requiring a PAM site. Similar to Cas12a, Cas14a can bind target nucleic acids and activate its ssDNA trans-cleavage activity, enabling its application in molecular detection of target nucleic acids. Unlike Cas12a, Cas14a only recognizes ssDNA targets. Therefore, amplified and enriched target nucleic acids need to be treated with T7 exonuclease. One of the amplification primers must carry phosphorothioate modification to ensure that T7 exonuclease only degrades one strand, leaving the ssDNA target strand for Cas14a-mediated molecular detection.
Cas14 enables high-fidelity SNP genotyping. Since DNA recognition does not rely on PAM sites, Cas14 has extremely stringent requirements for the accuracy of the recognized sequence. A single-base mismatch will severely inhibit its cleavage activity.
Components List:
| Component No. | Component Name | Appearance | 100pmol | 1000pmol | Storage | | C1540161A | Cas14a (10μM) | Liquid | 10μl | 100μl | -20℃. Avoid freeze/thaw cycle. | | C1540161B | Cas14a Diluent Buffer (1×) | Liquid | 100μl | 1ml | -20℃. Avoid freeze/thaw cycle. | | C1540161C | Cas14a Reaction Buffer (10×) | Liquid | 200μl | 2×1ml
| -20℃. Avoid freeze/thaw cycle. | | C1540161D | Positive Control | Liquid | 10μl | 10μl | -20℃. Avoid freeze/thaw cycle. |
* Cas14a Diluent Buffer (1×) also known as protein diluent. |
Cas14a sgRNA:
Cas14a sgRNA consists of tracrRNA, crRNA and Spacer. sgRNA binds to Cas14a to form a functional complex, which is specifically activated by the target sequence.
Cas14a sgRNA scaffold sequence (5'-3'):
CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAG UGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUC GAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAGAAAGUUGCAGAACCCGAAUAGACGAA
UGAAGGAAUGCAAC
sgRNA preparation is recommended using the CRISPR guide RNA in vitro transcription kit.
Assay Procedure
1. Prepare the Cas14a cleavage reaction system according to the table below. (Ice operation is recommended.)
| Component | Volume | Final Concentration |
|---|
| Cas14a Reaction Buffer (10×) | 2 μl | 1X | | Cas14a (10 μM)* | 0.5~2 μl | 250~1000 nM | | 10 μM sgRNA | 0.5~2 μl | 250~1000 nM | | 10 μM Target DNA** | X μl | - | | 10 μM ssDNA Reporter (for Cas14a)*** | 0.5~2 μl | 250~1000 nM | | Nuclease-free water | Up to 20 μl | - |
*Can be diluted with 1× Cas14a Dilution Buffer. Use immediately after dilution. **Target DNA can be ssDNA or dsDNA containing PAM sequence. The Positive Control consists of sgRNA, Reporter and ssDNA; add 5 μL into the 20 μL reaction system. (crRNA, Target DNA and ssDNA Reporter can be diluted with Nuclease-free Water. For extremely low-concentration Target DNA, dilution with 0.1% Tween 20 and low-binding tubes, pipette tips and other consumables are recommended.) *** Aladdin Reporter for Cas14a is recommended. (Cat#: R1540217, FAM-BHQ1 labeled ssDNA (Cas14a Fluorescent Reporter)). It is labeled with FAM fluorophore. |
2. Set the qPCR instrument at 37°C, and collect fluorescence signals every minute.
.png)
Precautions
1. If using a qPCR instrument, turn off the hot lid function or set the hot lid temperature to 42°C.
2. Cas14a cis-cleavage: Guided by sgRNA, Cas14a specifically cleaves target DNA. dsDNA targets require a PAM site, while ssDNA targets are PAM-independent.
3. Cas14a trans-cleavage: In the presence of target DNA, the Cas14a/sgRNA complex binds to target DNA, activating the trans-cleavage activity of Cas14a, which cleaves single-stranded DNA of any sequence in the reaction system.
4. When Cas14a binds to ssDNA targets, T7 exonuclease is required to digest amplified dsDNA targets to generate ssDNA targets for Cas14a-mediated molecular detection. (Note: One amplification primer needs phosphorothioate modification to ensure that T7 exonuclease only degrades the complementary strand, retaining the ssDNA target strand.)
5. Keep the experimental area clean. Wear clean gloves and masks during operation. Pipette tips, centrifuge tubes and other consumables used in the experiment must be RNase-free.
危险品化学品经营许可证（带存储）