SpCas9-NLS 是一种 RNA 介导的核酸内切酶，来源于化脓性链球菌 (Streptococcus pyogenes)。该酶可在 crRNA 和 tracrRNA( 或 两者连接形成的 sgRNA) 引导下，识别双链 DNA 靶标中 PAM 序列 (5'-NGG) 上游与 sgRNA spacer 序列互补的位置，并在 PAM 上游第 3 个碱基前端切割 DNA 双链。 SpCas9-NLS 常用于基因编辑，为提高细胞内编辑效率，本品 N 端带有猴病毒 40 (SV40) T 抗原核定位序列 (NLS)。本品亦可用于体外靶标 DNA 的剪切和基因克隆等。
组分表
| S1523005 | 组件 | 物理外观 | 100pmol | 1000pmol | 储存 | | S1523005A | SpCas9-NLS (10 μM) | Liquid | 10μl | 100μl | -20℃. Avoid freeze/thaw cycle. | | S1523005B | 10× Cut Buffer C | Liquid | 1ml | 1ml | -20℃. Avoid freeze/thaw cycle. |
|
失活条件
85℃温浴 5 min。
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%。
非特异性内切酶活性
将 10 pmol SpCas9-NLS 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。
DNase 活性
将 10 pmol SpCas9-NLS 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 活性
将 10 pmol SpCas9-NLS 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。
使用方法
体外顺式切割实验
① 在冰上配制如下反应体系：
| 试剂 | 使用量 | 终浓度 |
|---|
| 10× Cut Buffer C | 2 μl | 1× | | SpCas9-NLS (10 μM)ᵃ | 0.5 μl | 250 nM | | sgRNA (10 μM)ᵃ,ᵇ | 0.5 μl | 250 nM | | Target DNA (1 μM)ᵃ | 0.5 μl | 25 nM | | Nuclease-free water | To 20 μl | — |
|
a. 如果用凝胶电泳检测切割产物，建议 Target DNA 用量为 100~500 ng，长度 在 300 bp~3 kb 之间。建议 SpCas9-NLS : sgRNA : Target DNA 的摩尔比为 10:10:1，尽量确保靶标 DNA 被完全切割。
b. sgRNA 可参考以下序列设计：
5'-NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAGCAUA GCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC CGAGUCGGUG-3'（下划线表示 Target DNA 特异性互补配对的 spacer 序列 )。
② 37℃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。
常见问题
问题1： 观察到目的 DNA 切割不完全。
可能原因：
1. SpCas9 Nuclease、gRNA、target DNA 的比例不合适；
解决办法： 推荐 SpCas9 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。
2. 与 gRNA 的序列有关；
解决办法： 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。
3. gRNA 降解；
解决办法： 通过凝胶电泳验证 gRNA 的完整性。
3. 反应缓冲液不合适；
解决办法： 请使用 SpCas9 NLS 自带的缓冲液 10× Cut Buffer C。
问题2： 不同的 gRNA 之间的消化效率存在差异。
可能原因：
1. gRNA 的序列设计；
解决办法： 设计的 gRNA 需要进行序列与模板的验证。
2. gRNA 的质量；
解决办法： 利用琼脂糖凝胶电泳验证 gRNA 的完整性。
SpCas9-NLS is an RNA-guided endonuclease derived from Streptococcus pyogenes. Guided by crRNA and tracrRNA (or sgRNA formed by the two), this enzyme recognizes the position complementary to the sgRNA spacer sequence upstream of the PAM sequence (5'-NGG) in the double-stranded DNA target, and cleaves the DNA double strand just before the 3rd base upstream of the PAM. SpCas9-NLS is commonly used for gene editing. To improve intracellular editing efficiency, this product carries the nuclear localization signal (NLS) of the simian virus 40 (SV40) T antigen at the N-terminus. This product can also be used for in vitro target DNA cleavage and gene cloning, etc.
Components List
| S1523005 | Components | Appearance | 100pmol | 1000pmol | Storage | | S1523005A | SpCas9-NLS (10 μM) | Liquid | 10μl | 100μl | -20℃. Avoid freeze/thaw cycle. | | S1523005B | 10× Cut Buffer C | Liquid | 1ml | 1ml | -20℃. Avoid freeze/thaw cycle. |
|
Inactivation Condition
Incubate at 85℃ for 5 min.
Quality Control
- Protein PurityDetermined by SDS-PAGE, the protein purity is ≥95%.
- Non-specific Endonuclease Activity10 pmol of SpCas9-NLS 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.
- DNase Activity10 pmol of SpCas9-NLS 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 Activity10 pmol of SpCas9-NLS 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.
Protocol In Vitro Cis Cleavage Assay
① Prepare the following reaction system on ice:
| Reagent | Volume | Final Concentration |
|---|
| 10× Cut Buffer C | 2 μl | 1× | | SpCas9-NLS (10 μM)ᵃ | 0.5 μl | 250 nM | | sgRNA (10 μM)ᵃ,ᵇ | 0.5 μl | 250 nM | | Target DNA (1 μM)ᵃ | 0.5 μl | 25 nM | | Nuclease-free water | To 20 μl | — |
|
ᵃ. If the cleavage products are detected by gel electrophoresis, it is recommended to use 100~500 ng of Target DNA with a length between 300 bp and 3 kb. It is recommended that the molar ratio of SpCas9-NLS : sgRNA : Target DNA is 10:10:1 to ensure complete cleavage of the target DNA as much as possible.
ᵇ. sgRNA can be designed with reference to the following sequence:
5'-NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG-3' (The underlined part represents the spacer sequence complementary to the Target DNA specifically).
② Incubate at 37℃ for 30 min~1 h, then inactivate at 85℃ for 5 min.
③ Analyze the products by agarose gel electrophoresis.
Troubleshooting Problem
1: Incomplete cleavage of the target DNA is observed. Possible causes & solutions:
Improper ratio of SpCas9 Nuclease, gRNA, and target DNA.
- Solution: It is recommended that the molar ratio of SpCas9 Nuclease, gRNA, and target DNA is at least 10:10:1. The reaction can also be made more complete by appropriately extending the reaction time.
Related to the gRNA sequence.
- Solution: Select a more suitable gRNA sequence according to the target DNA. The efficiency of different gRNAs can vary greatly.
gRNA degradation.
- Solution: Verify the integrity of the gRNA by gel electrophoresis.
Inappropriate reaction buffer.
- Solution: Please use the 10× Cut Buffer C provided with SpCas9 NLS.
Problem 2: Differences in digestion efficiency between different gRNAs. Possible causes & solutions:
gRNA sequence design.
- Solution: The designed gRNA needs to be verified against the template sequence.
gRNA quality.
- Solution: Verify the integrity of the gRNA by agarose gel electrophoresis.
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