Premade Cas9 Protein
VectorBuilder offers ready-to-use Cas9 proteins that can be used for direct delivery in gene editing experiments, including knockout (KO) and knockin, with high efficiency and lower risk of off-target effects. Alternative Cas9 variants and other Cas family proteins are available through our recombinant protein production service.
Highlights:
- Cas9 proteins isolated from E. coli or insect cells and optimized for high editing performance.
- High efficiency for both direct protein transfection and RNP complex formation with your gRNA of choice.
- Comprehensive and strict quality control to ensure high purity and optimal functionality.
Ordering Information Price Match
Shipping and storage
SpCas9 (E. coli) is shipped as a lyophilized powder at ambient temperature, or on dry ice if bundled with temperature-sensitive items. Upon receipt, store the product at –20°C to –80°C under sterile conditions. Once reconstituted, aliquot the protein immediately to avoid freeze-thaw cycles. When stored under sterile conditions at –20°C to –80°C, the product remains stable for up to one year.
SpCas9 (Insect Cells) is shipped as a liquid on dry ice; note that dry ice may incur additional charges. For a more cost-effective alternative, SpCas9 (E. coli) is available for shipping at ambient temperature. Upon receipt, aliquot the protein immediately to avoid freeze-thaw cycles. When stored under sterile conditions at –20°C to –80°C, the product remains stable for up to one year.
Experimental Validation
Experimental characterization
CRISPR-mediated gene knockout
CRISPR-mediated gene knockin
Characterization of purified SpCas9 protein produced from bacterial and insect cells.
- E. coli
- Insect cells
Figure 1. Characterization of the purified SpCas9 protein produced with the E. coli system. (A) SDS-PAGE analysis showed the molecular mass of the purified protein to be approximately 139.7 kDa (under reducing conditions), and the purity to be ≥95%. (B) The purity was confirmed to be ≥95% by SEC-HPLC. (C) Biological activity of the purified Cas9 protein was assessed using an in vitro DNA cleavage assay. A 760 bp linear DNA template and gRNA were combined with Cas9 (+) or no enzyme (−). The addition of Cas9 resulted in the predicted cleavage of the substrate into two DNA fragments (450 and 310 bp). VectorBuilder’s SpCas9 achieved ≥90% substrate cleavage. N: negative control; VB: VectorBuilder SpCas9 (E. coli).
Figure 2. Characterization of the purified SpCas9 protein produced with the baculovirus-insect system. (A) SDS-PAGE analysis shows the molecular mass of the purified protein to be approximately 139.5 kDa (under reducing conditions), and the purity to be ≥90%. (B) The purity was determined to be ≥95% by SEC-HPLC. (C) Biological activity of the purified Cas9 protein was assessed using an in vitro DNA cleavage assay. A 760 bp linear DNA template and gRNA were combined with Cas9 (+) or no enzyme (−). The addition of Cas9 resulted in the predicted cleavage of the substrate into two DNA fragments (450 and 310 bp). VectorBuilder’s SpCas9 achieved ≥90% substrate cleavage. N: negative control; VB: VectorBuilder SpCas9 (Insect Cells).
Figure 3. Generating homozygous KO mutants using the gRNA/Cas9 RNP approach with dual gRNAs for deletion of a DNA fragment. (A) The editing RNP is electroporated into target cells with two sites on the targeted gene to delete a 13 kb region, and single clones are isolated and screened. The genotypes of the candidates are validated using PCR and Sanger sequencing. (B) Four primers, P1 to P4, are used in three PCRs to differentiate KO and WT clones. Based on the PCR results (C), clone 1 is validated to be homozygous KO mutant, which is also confirmed by sequencing results (D).
Figure 4. CRISPR-mediated gene knockin in iPSCs. Knockin of UBC-driven EGFP (2432 bp) into iPSCs was achieved by electroporation of Cas9/gRNA RNP complex and donor vector. (A) Confirmation of EGFP knockin at target site by Sanger sequencing. (B) Genotyping PCR of four single clones with homozygous knockin. The WT locus is 762 bp and the locus with EGFP knockin is 3194 bp. (C) EGFP fluorescence in knockin cells by microscopy. (D) Karyotyping results. (E) Expression of pluripotency markers NANOG, OCT4, and SOX2 in EGFP knockin iPSCs by immunofluorescence to confirm maintained pluripotency.
Resources
FAQ
Cas9 RNPs offer several important advantages over plasmid- or virus-based delivery systems. Because the Cas9 protein and gRNA are introduced in their active forms, gene editing occurs rapidly without the need for transcription or translation. In addition, delivering only protein and RNA means there is no risk of random insertion of DNA into the host genome, and off-target cuts by Cas9 are minimized compared to other delivery approaches because of the speed at which the protein is degraded by the host cell. Additionally, RNPs can be delivered efficiently by electroporation, making them suitable for many cells that are difficult to chemically transfect. This approach also works independently of cell-type specific promoter activity, making it effective across a wide range of cell types.
The transient nature of Cas9 RNPs means that editing occurs quickly after electroporation and then tapers off as the complex is degraded in the cell. This can be useful when transient Cas9 activity is desired, for example, to reduce long-term off-target effects. However, it also means that experiments must be carefully timed, since most editing events will happen relatively quickly after delivery rather than being spread over many days as with more stable Cas9 delivery systems.
