阿拉丁生产的 Cas9 NLS (SpCas9-NLS)，即含有核定位信号的 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。本产品可以用于细胞 内的 CRISPR/Cas9 系统介导的基因编辑，也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。Cas9 NLS 在 Cas9 蛋白的 N 端和 C 端都含有 SV40 T 抗原的核定位序列(Nuclear localization signal or nuclear localization sequence, NLS)，使 Cas9 与 gRNA 形成的复合物在转染进入细胞后、能迅速地从细胞质进入到细胞核内，从而大大地提高 了基因编辑的效率。Cas9 NLS 可以通过显微注射、电穿孔和脂质体介导等方法进入细胞，而这种不需要使用 DNA 的系统不会产生外源 DNA 整合 至细胞基因组的风险 。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/Cas9 系统由 Cas9 Nuclease 和 gRNA 复 合物所组成。gRNA，也称 sgRNA (single guide RNA)，由 18-20bp 与靶基因序列互补的 CRISPR RNA (crRNA)序列以及能与 Cas9 特异性结合的 trans-activating crRNA (tracrRNA)序列组成。
组分和说明
| C744421 | 组分名称
| 500pmol | 5×500pmol | 储存
|
| C744421A | Cas9 NLS (20μM) | 25µl | 5×25µl | -20℃. 避免反复冻融。 |
| C744421B | Cas9 NLS Reaction Buffer (10×) | 2ml | 5×2ml | -20℃. 避免反复冻融。 |
产品应用
细胞基因编辑、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
CRISPR/Cas9 是一项突破性的基因组编辑技术，操作便捷，应用广泛。
使用说明
1.Cas9-gRNA 复合物电转细胞(以 Neon® 电转系统为例)。
a.所需实验材料：
(a)HEK293 细胞系，若使用其他细胞如下实验方法可能需要进行适当优化
(b)Cas9 NLS
(c)特异性靶向目的基因的 gRNA
(d)Neon Transfection System 10μl kit (Thermo)
(e) PBS
(f)胰酶细胞消化液
(g)含有 10%FBS 以及 Glutamax 的 DMEM
(h)24 孔细胞培养板，或者实验需要使用到的其它规格的培养板
(i)基因组编辑突变检测试剂盒
b.电转实验步骤：
(a)在电转前一天(18-24h)接种细胞(具体的细胞数量据细胞类型、大小和细胞生长速度等而定)，使第二天细胞密度达到约 70-90%。
(b)以 24 孔板为例，参考下表设置 Cas9-gRNA 形成反应。Resuspension Buffer R 包括在 Neon transfection kit 中，在此步骤中，使 用 Resuspension Buffer R，而无需使用 Cas9 NLS 附带的 Reaction Buffer (10×)。
| Reagent | Volume |
|---|
| Resuspension Buffer R | 10 μl |
| Cas9 NLS (20 μM) | 2.5 μl |
| gRNA (50 μM) | 2 μl |
| Total Volume | 14.5 μl |
(c)轻轻混匀上述反应体系，并在室温下孵育 20 分钟。
(d)在孵育过程中，用胰酶消化细胞，将细胞重悬于 5-10ml 完全培养液中，然后洗涤一次以除去胰蛋白酶残留。细胞计数确定活细胞数 量。
(e)计算整个实验所需的细胞数量，每次转染需要 1-2×10⁵细胞，将细胞加入到无菌的离心管中。在 500×g 离心 5min。用 PBS 洗涤细胞 一次。
(f)根据细胞数量计算 Resuspension Buffer R 的体积，每次转染需要 10.5μl Resuspension Buffer R。用相应体积的 Resuspension Buffer R 重悬细胞。
(g)24 孔板中每孔加入 500μl 完全培养基。
(h)每 14.5μl 的 Cas9-gRNA 体系中轻柔加入 10.5μl 细胞悬液并混匀。
(i)将 10μl Cas9-gRNA 与细胞混合物添加到 10μl 的 Neon 移液器吸头腔内。按照以下条件进行细胞电转：1700V，20ms，1pulse。(以 上条件仅供参考，具体操作步骤及条件请参考您使用的电转试剂盒说明书进行)
(j)立即将细胞转移到含有培养基的 24 孔板中。
(k)将细胞在培养箱中孵育 48-72h。
(l)使用基因组编辑突变检测试剂盒对电转后的细胞进行检测，具体步骤参照产品说明书。
2.Cas9 NLS 体外消化 DNA。
a.溶解并混匀体外消化反应所需的各种溶液。将 Cas9 NLS、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。用无核酸酶水稀释适量 Cas9 NLS Reaction Buffer (10×)至 Cas9 NLS Reaction Buffer (1×)。Cas9 NLS (20μM)推荐使用 Cas9 NLS Reaction Buffer (1×)稀释适量备用，稀释后宜尽快使用，不宜冻存后使用，以避免反复冻融导致酶活性下降。
b.按照下表配制反应体系(以 30μl 体系为例)：
| Reagent | Volume |
| Water (DNase/RNase-Free) | 20µl |
| Cas9 NLS Reaction Buffer (10×) | 3µl |
| gRNA (300nM) | 3µl |
| Cas9 NLS (1μM) | 1µl |
| Total Reaction Volume | 27µl |
c.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。
d.加入 3μl 30nM 底物 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。
e.每个样品中加入 1μl 蛋白酶 K ，轻轻混匀， 室温孵育 10min。
f.每个反应体系中加入 6μl DNA 上样缓冲液(6×)，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。 通过体外的 Cas9 酶切实验，可以判断所设计的 gRNA 的效果是否理想或者优化筛选出理想的 gRNA 用于细胞或动物实验。
3.Cas9 NLS 也可以通过适当的蛋白转染试剂把 Cas9-gRNA 转染至细胞内，具体请参考相应的 蛋白转染试剂的产品说明书。
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、gRNA、target DNA 的比例不合适引起的，推荐 Cas9 Nuclease、gRNA、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 gRNA 的序列有关，可以根据 target DNA 选择更合适的 gRNA 序列，不同的 gRNA 的效果会差别比较大。
c.可能由于 gRNA 降解引起的，可以通过凝胶电泳验证 gRNA 的完整性。
d.反应缓冲液可能不合适，请使用 Cas9 NLS 自带的缓冲液 Cas9 NLS Reaction Buffer (10×)。
2.为什么不同的 gRNA 之间的消化效率存在差异?
a.gRNA 的序列设计可能会影响消化效率，所设计的 gRNA 需要进行序列与模板的验证。
b.gRNA 的质量也可能会影响消化效率，利用琼脂糖凝胶电泳验证 gRNA 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，可以使用 RNase and DNase Away 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。
Cas9 NLS (SpCas9-NLS) produced by our company, namely CRISPR-associated endonuclease Cas9 with nuclear localization signal (also known as Csn1), is a sequence-specific double-stranded DNA endonuclease derived from Streptococcus pyogenes. It is expressed and purified on our self-developed technical platform and can cleave double-stranded DNA under the guidance of gRNA. This product can be applied for CRISPR/Cas9-mediated gene editing in cells, in vitro screening of high-efficiency guide RNA (gRNA) sequences, gRNA-directed cleavage of specific DNA sequences, linearization of double-stranded circular DNA containing specific sequences, and other applications. Cas9 NLS carries SV40 T-antigen nuclear localization sequences (NLS) at both the N-terminus and C-terminus of Cas9 protein. After transfection into cells, the Cas9-gRNA complex can rapidly translocate from the cytoplasm into the cell nucleus, which greatly improves gene editing efficiency. Cas9 NLS can be delivered into cells via microinjection, electroporation and liposome-mediated methods. This DNA-free system eliminates the risk of exogenous DNA integration into the cellular genome. CRISPR/Cas9 is a breakthrough genome editing technology with convenient operation and wide applications. The CRISPR/Cas9 system consists of Cas9 Nuclease and gRNA complex. gRNA, also referred to as sgRNA (single guide RNA), is composed of an 18–20 bp CRISPR RNA (crRNA) sequence complementary to the target gene and a trans-activating crRNA (tracrRNA) sequence that specifically binds Cas9.
Components List
| Component No. | Component Name | 500pmol | 5×500pmol
| Storage |
| C744421A | Cas9 NLS (20 μM)
| 25μl | 5×25μl
| -20℃. Avoid freeze/thaw cycle.
|
| C744421B | Cas9 NLS Reaction Buffer (10×)
| 2ml | 5×2ml
| -20℃. Avoid freeze/thaw cycle.
|
Applications
Cellular gene editing, 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
CRISPR/Cas9 is a breakthrough genome editing technology with convenient operation and wide applications.
Protocol
1. Electroporation of Cas9-gRNA complex into cells (example with Neon® Transfection System)
a. Required materials:
(a) HEK293 cell line; optimization may be required for other cell types.
(b) Cas9 NLS
(c) gRNA specifically targeting the gene of interest
(d) Neon Transfection System 10 μl kit (Thermo)
(e) PBS
(f) Trypsin cell dissociation solution
(g) DMEM supplemented with 10% FBS and Glutamax
(h) 24-well cell culture plate or plates of other specifications as needed
(i) Genome editing mutation detection kit
b. Electroporation procedure:
(a) Seed cells 18–24 h before electroporation. The cell number depends on cell type, cell size and growth rate, to reach 70–90% confluence on the next day. (b) Take 24-well plate as example, prepare Cas9-gRNA complex reaction as the table below. Resuspension Buffer R is included in Neon transfection kit. Use Resuspension Buffer R for this step instead of the supplied Cas9 NLS Reaction Buffer (10×).
| Reagent | Volume |
|---|
| Resuspension Buffer R | 10 μl |
| Cas9 NLS (20 μM) | 2.5 μl |
| gRNA (50 μM) | 2 μl |
| Total Volume | 14.5 μl |
(c) Gently mix the reaction mixture and incubate at room temperature for 20 min.
(d) During incubation, trypsinize cells, resuspend cells in 5–10 ml complete medium, wash once to remove residual trypsin, and count viable cells.
(e) Calculate total cell number for the experiment. Each transfection requires 1–2 × 10⁵ cells. Transfer cells to sterile centrifuge tubes, centrifuge at 500×g for 5 min, and wash once with PBS.
(f) Calculate the volume of Resuspension Buffer R according to cell number. Each transfection requires 10.5 μl Resuspension Buffer R. Resuspend cells with corresponding volume of Resuspension Buffer R.
(g) Add 500 μl complete medium per well of 24-well plate.
(h) Gently add 10.5 μl cell suspension into 14.5 μl Cas9-gRNA mixture and mix well.
(i) Load 10 μl Cas9-gRNA/cell mixture into 10 μl Neon pipette tip. Perform electroporation under parameters: 1700 V, 20 ms, 1 pulse. (The parameters are for reference only; refer to the manual of your electroporation kit for detailed operation and conditions.)
(j) Immediately transfer cells into 24-well plate containing medium.
(k) Incubate cells in incubator for 48–72 h.
(l) Detect gene editing mutation of electroporated cells using genome editing mutation detection kit, follow the product manual for detailed steps.
2. In vitro DNA digestion with Cas9 NLS
a. Prepare and mix all solutions for in vitro digestion. Place Cas9 NLS, gRNA and substrate DNA on ice. Dilute gRNA to 300 nM and substrate DNA to 30 nM with nuclease-free water. Dilute Cas9 NLS Reaction Buffer (10×) to 1× with nuclease-free water. It is recommended to dilute Cas9 NLS (20 μM) with 1× Cas9 NLS Reaction Buffer for immediate use. Do not freeze diluted enzyme to avoid activity loss caused by repeated freeze-thaw cycles.
b. Prepare reaction system (30 μl final volume as example):
| Reagent | Volume |
|---|
| Water (DNase/RNase-Free) | 20 μl |
| Cas9 NLS Reaction Buffer (10×) | 3 μl |
| gRNA (300 nM) | 3 μl |
| Cas9 NLS (1 μM) | 1 μl |
| Total Reaction Volume | 27 μl |
c. Gently pipette or vortex briefly to mix, spin down briefly at room temperature to collect liquid at tube bottom. Pre-incubate at 25℃ for 10 min.
d. Add 3 μl of 30 nM substrate DNA (30 μl final volume), mix gently, spin down. Incubate at 37℃ for 15 min. Reaction time can be extended appropriately to 30–120 min as required.
e. Add 1 μl Proteinase K to each sample, mix gently, incubate at room temperature for 10 min.
f. Add 6 μl 6× DNA loading buffer to each reaction, analyze by agarose gel electrophoresis of appropriate concentration. Samples can be stored at -20℃ if electrophoresis is not performed immediately.
The in vitro Cas9 cleavage assay can evaluate the performance of designed gRNA, or screen and optimize ideal gRNA for cellular or animal experiments.
3. Cas9 NLS can also be delivered into cells together with gRNA via suitable protein transfection reagents. Please refer to the manual of corresponding protein transfection reagent for details.
Frequently Asked Questions
1. Why is incomplete cleavage of target DNA observed?
a. Improper molar ratio of Cas9 Nuclease, gRNA and target DNA. Recommended molar ratio ≥10:10:1. Reaction can be extended to achieve complete digestion.
b. gRNA sequence affects efficiency. Select appropriate gRNA for target DNA; cleavage efficiency varies significantly between different gRNAs.
c. Degradation of gRNA. Verify gRNA integrity by gel electrophoresis.
d. Inappropriate reaction buffer. Use the supplied Cas9 NLS Reaction Buffer (10×).
2. Why are digestion efficiencies different between various gRNAs?
a. gRNA sequence design affects digestion efficiency. Validation between gRNA sequence and template is required.
b. gRNA quality impacts digestion efficiency. Verify gRNA integrity by agarose gel electrophoresis.
Precautions
(1) Operations involving gRNA and DNA require RNase-free and DNase-free environment. All self-prepared reagents and consumables shall be nuclease-free. For potential nuclease contamination, treat solution with 0.01% DEPC overnight followed by autoclaving. Disposable mask is recommended during operation.
(2) To remove nucleases on bench surface and equipment, RNase and DNase Away can be used. RNase inhibitor is recommended in reaction system to protect RNA from degradation.
(3) This product is for research use only by qualified professionals. Not for clinical diagnosis or therapy, not for food or pharmaceutical use. Do not store in residential premises.
(4) Wear lab coat and disposable gloves for personal safety.
危险品化学品经营许可证（带存储）