| Product Description | Aladdin's Cas9 NLS (SpCas9-NLS) is a Streptococcus Pyogenes Cas9 Nuclease with a nuclear localization signal, recombinantly expressed in E.coli and purified using the PerfectProtein™ technology platform developed by aladdin. It is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a guide RNA (gRNA) loaded into the protein. This product can be used for intracellular CRISPR/Cas9-mediated gene editing, in vitro screening of efficient gRNA sequences, gRNA-guided cleavage of specific DNA sequences, and linearization of double-stranded circular DNA containing particular sequences, etc.Cas9 NLS contains the nuclear localization signal or sequence (NLS) of SV40 T antigen at both N and C termini, which enables the Cas9-gRNAcomplex to be imported into cell nucleus rapidly after transfection and improves the gene editing efficiency significantly. Cas9-gRNA complex without any DNA molecules can be transfected into cells by microinjection, electroporation, and liposome-mediated methods, thus eliminating the risk of exogenous DNA integration into the cellular genome [1].CRISPR/Cas9-based genome editing technology is a new breakthrough, which has been widely used in genome engineering. CRISPR (clustered regularly interspaced short palindromic Repeats) is an adaptive immune system for gene silencing of exogenous phage or viral nucleic acid by using RNA-guided DNA nuclease Cas9, on which the CRISPR/Cas9-based gene-editing technique has been developed to be widely used in prokaryotes and eukaryotes. This technology enables site-specific cleavage of targeted genomic DNA by gRNA-guided Cas9 and introduction of frameshift mutation at the cutting site by error-prone repair or homologous recombination, during which gRNA ensures the specificity of the cleavage site. The CRISPR technology can not only achieve gene knockout, but also achieve a variety of mutations such as point mutation and insertion mutation, especially in the clinical application can be used to repair undesirable mutations [2, 3]. Moreover, transcriptional activation or inhibition of target genes can be achieved by recruiting transcriptional activation or transcriptional directly or indirectly via the Cas9 mutant without endonuclease activity (dCas9).CRISPR/Cas9 system is composed of Cas9 Nuclease and gRNA. The gRNA consists of two parts
Application： In vitro screening of highly efficient gRNA, gene editing, gRNA-guided cleavage of specific double-stranded DNA, selective linearization of double-stranded DNA containing particular sequences, etc.SpCas9 Purity: Free of RNase, DNA exonuclease, and non-gRNA-dependent DNA endonuclease.SpCas9 Concentration: 20μM (~3.2μg/μl)。SpCas9 Storage Buffer: 10mM Tris, 300mM NaCl, 0.1mM EDTA,1mM DTT, 50% (v/v) Glycerol, pH7.4 at 25℃。SpCas9 Reaction Buffer (10X): 500mM Tris-HCl, 1M NaCl, 100mM MgCl2, 1mg/ml BSA, pH7.9 at 25℃。
Source： Recombinant spCas9 expressed and purified from E. coli.
Reaction Buffer： 500mM Tris-HCl, 1M NaCl, 100mM MgCl2, 1mg/ml BSA, pH7.9@25℃。
Precautions： We strongly recommend wearing a disposable mask and using nuclease-free tubes and reagents to avoid RNase contamination.We recommend using DNase and DNase Away (, R0123) to remove nucleases on the surface of benches, pipettes, or other equipment. RNase Inhibitor can also be used to protect RNA from degradation.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： 1. Electrotransformation of the Cas9-gRNA complex into cells (use the Neon® Transfection System as an example)a. Materials required(a) HEK293 cell line. For other cell lines, the following protocol should be properly optimized.(b) Cas9 NLS(c) gRNA for target genes(d) Neon Transfection System 10μl kit (Thermo) (e) PBS ( Ultrapure Water which is sterile and DNase/RNase-free. b. Set up the reaction in a microfuge tube on ice as follows: ReagentVolume Nuclease-free Water 20µl Cas9 NLS Reaction Buffer (10X) 3µl gRNA (300nM) 3µl Cas9 NLS (1μM) 1µl Total Reaction Volume 27µl c. Mix well gently by pipetting or vortex. Pulse-spin in a microfuge and incubate the reaction at 25℃ for 10 minutes. d. Add 3µl of 30nM substrate DNA, mix gently, and pulse-spin in a microfuge. Incubate at 35℃ for 15 minutes. If necessary, prolong the incubation time to 30-120 minutes. e. Add 3µl of Proteinase K (, ST532). Mix gently and incubate at room temperature (~25℃) for 10 minutes.f. Examine the reaction products by agarose gel electrophoresis after mixing with the 6X DNA loading buffer (, D0071). The reaction products can also be stored at -20℃ for future analysis. The In vitro Cas9-gRNA cleavage assay on substrate DNA can be used to determine whether the designed gRNA works well for target DNA. 3. Cas9-gRNA can also be transfected into cells with appropriate protein transfection reagents. please refer to manufacture's instructions for the transfection.FAQ:1. The substrate DNA is digested incompletely.a. Incomplete digestion may be caused by an incorrect ratio of Cas9 NLS, gRNA, and target DNA. For complete digestion, we recommend a 10:10:1 molar ratio of Cas9 NLS:gRNA:target DNA. The reaction time can also be extended.b. Incomplete digestion may be caused by inappropriate gRNA sequences. Redesign the gRNA sequence.c. Incomplete digestion may be caused by the degradation of gRNA. Check the integrity of the gRNA by gel electrophoresis.d. Incomplete digestion may be caused by inappropriate buffer. Please use Cas9 NLS Reaction Buffer (10X) provided in this product.2. Why is there a difference in cleavage efficiency between different gRNAs?a. The sequence of gRNAaffects the cleavage efficiency, which should be validated in vitro before transfection.b. The quality of gRNA may also affect the cleavage efficiency. Check the integrity of the gRNA by gel electrophoresis.
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