本公司生产的 Cas9 Nuclease (SpCas9)，即 CRISPR-associated endonuclease Cas9 也称 Csn1，是本公司自主研发的技术平台表达、纯化获得 的一种来源于 Streptococcus pyogenes，能在 gRNA 引导下序列特异性地切割双链 DNA 的核酸内切酶。本产品可以用于体外筛选高效的 guide RNA (gRNA)序列、特定 DNA 序列在 gRNA 引导下的剪切、含有特定序列的双链环形 DNA 的线性化等用途。CRISPR/Cas9 是一项突破性的基因组 编辑技术，操作便捷，应用广泛。CRISPR (clustered regularly interspaced short palindromic repeats)是一种原核生物利用 RNA 引导的 DNA 核酸酶 Cas9 对外源的噬菌体或病毒核酸进行基因沉默的获得性免疫系统(adaptive immune system)，后续在此基础上逐渐发展为广泛应用于原 核和真核生物的越来越成熟的基因编辑技术。该技术能够在 gRNA 引导下通过 Cas9 对原核和真核生物的基因组 DNA 的靶向序列进行位点特异性 的切割，然后通过易错修复(error-prone repair)或同源重组(homologous recombination)在切割位点改变或插入序列来产生移码突变，从而通过 基因编辑而实现基因敲除。其中 gRNA 确保识别位点的特异性。随着 CRISPR 技术的发展，该技术目前不仅可以实现基因敲除，还可以实现基因的 点突变、插入突变等多种突变方式，特别是在临床应用方面可以用于修复不良突变等。同时通过构建没有内切酶活性的 Cas9 突变体 dCas9，通过 与 dCas9 直接融合表达或间接招募转录激活或转录抑制因子，可以实现 sgRNA 靶向基因的转录激活或转录抑制。
纯度（Purity）：不含 DNA 外切酶，不含非 gRNA 依赖的 DNA 内切酶，不含 RNA 酶。
组分和说明
| C744422 | Component | 50pmol | 5*50pmol | 20*50pmol | Storage | | C744422A | Cas9 Nuclease (1μM) | 50µl | 5*50µl | 20*50µl | -20℃. Avoid freeze/thaw cycle. | | C744422B | Cas9 Reaction Buffer (10X) | 0.2ml | 5*0.2ml | 20*0.2ml | -20℃. Avoid freeze/thaw cycle. |
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产品应用
体外筛选高效 gRNA 序列、特定双链 DNA 在 gRNA 引导下的剪切、含有特定序列双链 DNA 的选择性线性化等。
产品优势
Cas9 Nuclease 可与向导 RNA（gRNA）形成稳定的核糖核蛋白（RNP）复合物，进入细胞核后实现高效的基因组 DNA 切割。 在多种细胞系（包括标准细胞系、免疫细胞、原代细胞和干细胞）中表现出稳定的高编辑效率。
使用说明
1.溶解并混匀体外消化反应所需的各种溶液。将 Cas9 Nuclease、gRNA、底物 DNA 置于冰浴上，使用无核酸酶水稀释 gRNA 至 300nM， 底物 DNA 至 30nM。
2.按照下表配制反应体系(以 30μl 体系为例)：
| Reagent | Volume | | Nuclease-free Water | 20µl | | Cas9 Reaction Buffer (10X) | 3µl | | gRNA (300nM) | 3µl | | Cas9 Nuclease (1μM) | 1µl | | Total Reaction Volume | 27µl |
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3.用移液器轻轻吹打混匀或轻微 Vortex 混匀，室温离心数秒，使液体积聚于管底。25℃预孵育 10min。
4.加入 3μl 30nM 底物靶 DNA (30μl 最终体积)，轻轻混匀(用移液器轻轻吹打混匀或用涡旋混合器在最低速度轻轻混匀)，随后离心沉淀 液体，37℃孵育 15min，反应时间可以根据实际情况适当延长至例如 30-120min。
5.每个样品中加入 1μl 蛋白酶 K，轻轻混匀， 室温孵育 10min。
6.每个反应体系中加入 6μl DNA 上样缓冲液(6X)，然后使用适当浓度的琼脂糖凝胶进行电泳分析。如果不立即电泳，可以-20℃保存备用。
常见问题：
1.为什么观察到目的 DNA 切割不完全? a.可能是由于 Cas9 Nuclease、sgRNA、target DNA 的比例不合适引起的，推荐 Cas9 Nuclease、sgRNA、target DNA 的摩尔比例至 少为 10:10:1。也可以通过适当延长反应时间使反应更加充分。
b.可能与 sgRNA 的序列有关，可以根据 target DNA 选择更合适的 sgRNA 序列，不同的 sgRNA 的效果会差别比较大。
c.可能由于 sgRNA 降解引起的，可以通过凝胶电泳验证 sgRNA 的完整性。
保存条件：
-20℃保存，≤0℃运输
注意事项：
（1）本产品使用时会涉及 gRNA 和 DNA 的操作，必须注意 RNase-free 和 DNase-free 的相关操作。所有自行准备的试剂和耗材也都应是 Nuclease-free 的。如果可能有核酸酶污染，可考虑用 0.01%的 DEPC 处理过夜，然后高温高压处理后使用。操作时建议戴一次性口罩操作。
（2）对于操作环境中核酸酶的去除，推荐使用阿拉丁生产的 RNase and DNase Away (R749971) 以去除实验桌、仪器设备等表面或其它接触面上的 核酸酶。反应体系中推荐加入 RNase Inhibitor 以保护 RNA 不被降解。
（3）本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
（4）为了您的安全和健康，请穿实验服并戴一次性手套操作。
The Cas9 Nuclease (SpCas9) produced by our company—also known as CRISPR-associated endonuclease Cas9 or Csn1—is an endonuclease derived from Streptococcus pyogenes. It is expressed and purified through our independently developed technology platform, and can sequence-specifically cleave double-stranded DNA under the guidance of gRNA.This product is applicable for multiple uses, including in vitro screening of high-efficiency guide RNA (gRNA) sequences, gRNA-directed cleavage of specific DNA sequences, and linearization of double-stranded circular DNA containing target sequences.CRISPR/Cas9 is a breakthrough genome editing technology with simple operation and wide applications. CRISPR (clustered regularly interspaced short palindromic repeats) is an adaptive immune system in prokaryotes. It uses the RNA-guided DNA nuclease Cas9 to silence foreign phage or viral nucleic acids. Subsequently, it has evolved into an increasingly mature gene editing technology widely used in both prokaryotes and eukaryotes.The technology enables site-specific cleavage of target sequences in the genomic DNA of prokaryotes and eukaryotes via Cas9 under gRNA guidance. Frameshift mutations are then generated by altering or inserting sequences at the cleavage site through error-prone repair or homologous recombination, thereby achieving gene knockout through gene editing. Among these, gRNA ensures the specificity of the recognition site.With the development of CRISPR technology, it now supports multiple mutation types beyond gene knockout, such as gene point mutations and insertion mutations. It is particularly useful in clinical applications, including the repair of harmful mutations. Additionally, by constructing dCas9—a Cas9 mutant without endonuclease activity—transcriptional activation or inhibition of sgRNA-targeted genes can be achieved through direct fusion expression with dCas9 or indirect recruitment of transcriptional activators or repressors.
Purity: Free of exonuclease, non-gRNA-dependent endonuclease, and RNase.
| C744422 | Component | 50pmol | 5*50pmol | 20*50pmol | Storage | | C744422A | Cas9 Nuclease (1μM) | 50µl | 5*50µl | 20*50µl | -20℃. Avoid freeze/thaw cycle. | | C744422B | Cas9 Reaction Buffer (10X) | 0.2ml | 5*0.2ml | 20*0.2ml | -20℃. Avoid freeze/thaw cycle. |
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Product Applications
In vitro screening of high-efficiency gRNA sequences, gRNA-guided cleavage of specific double-stranded DNA, selective linearization of double-stranded DNA containing specific sequences, etc.
Product Advantages
Cas9 Nuclease can form a stable ribonucleoprotein (RNP) complex with guide RNA (gRNA), which enters the nucleus to achieve efficient genomic DNA cleavage. It exhibits stable and high editing efficiency in various cell lines, including standard cell lines, immune cells, primary cells and stem cells.
Usage Instructions
1. Dissolve and mix all solutions required for in vitro digestion reactions. Place Cas9 Nuclease, gRNA and substrate DNA on ice. Dilute gRNA to 300nM and substrate DNA to 30nM using nuclease-free water.
2. Prepare the reaction system according to the following table (taking 30μl system as an example):
| Reagent | Volume | | Nuclease-free Water | 20µl | | Cas9 Reaction Buffer (10X) | 3µl | | gRNA (300nM) | 3µl | | Cas9 Nuclease (1μM) | 1µl | | Total Reaction Volume | 27µl |
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3. Gently mix by pipetting up and down or slight vortexing. Centrifuge briefly at room temperature to collect the liquid at the bottom of the tube. Pre-incubate at 25℃ for 10 minutes.
4. Add 3μl of 30nM substrate target DNA (final volume 30μl). Mix gently (by pipetting up and down or light vortexing at the lowest speed), then centrifuge to pellet the liquid. Incubate at 37℃ for 15 minutes; the reaction time can be appropriately extended to 30-120 minutes depending on actual conditions.
5. Add 1μl of Proteinase K to each sample, mix gently, and incubate at room temperature for 10 minutes.
6. Add 6μl of 6X DNA Loading Buffer to each reaction system, then perform electrophoresis analysis using an appropriate concentration of agarose gel. If electrophoresis is not performed immediately, the sample can be stored at -20℃ for later use.
Frequently Asked Questions
1. Why is the target DNA cleavage incomplete?
a. It may be caused by an inappropriate molar ratio of Cas9 Nuclease, sgRNA, and target DNA. A molar ratio of at least 10:10:1 for Cas9 Nuclease:sgRNA:target DNA is recommended. The reaction can also be made more complete by appropriately extending the reaction time.
b. It may be related to the sgRNA sequence. A more suitable sgRNA sequence can be selected based on the target DNA, as the efficiency of different sgRNAs varies significantly.
c. It may be caused by sgRNA degradation. The integrity of sgRNA can be verified by gel electrophoresis.
Storage Conditions
Store at -20℃ and transport at ≤0℃.
Precautions
(1) The use of this product involves operations with gRNA and DNA, so strict RNase-free and DNase-free practices must be followed. All self-prepared reagents and consumables should also be Nuclease-free. If nuclease contamination is possible, treat with 0.01% DEPC overnight, followed by autoclaving before use. It is recommended to wear a disposable mask during operation.(2) For the removal of nucleases in the operating environment, Aladdin's RNase and DNase Away (R749971) is recommended to eliminate nucleases on surfaces such as laboratory benches, instruments, and other contact surfaces. Adding RNase Inhibitor to the reaction system is recommended to protect RNA from degradation.
(3) This product is for scientific research use only by professional personnel. It shall not be used for clinical diagnosis or treatment, food or pharmaceuticals, nor stored in ordinary residences.(4) For your safety and health, wear a lab coat and disposable gloves during operation.
ZJ26F0131403
危险品化学品经营许可证（带存储）