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. |
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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 | — |
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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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