Cell Engineering icon

XactEdit™ Cas9 Nuclease with NLS

Perform targeted guide RNA-directed double-stranded DNA cleavage using this highly purified enzyme. XactEdit™ Cas9 Nuclease with NLS is a purified recombinant Streptococcus pyogenes Cas9 protein containing a nuclear localization signal (NLS). When complexed with an appropriately designed guide RNA (gRNA), XactEdit™ Cas9 Nuclease mediates site-specific, targeted double-stranded DNA cleavage. The introduction of Cas9:gRNA complexes (RNPs) into cells creates double-stranded DNA breaks (DSBs) which have been used for a variety of targeted genome-editing studies, such as homologous knock-in and gene knock-out.

Available as a kit or as a standalone enzyme at a concentration of either 1 mg/mL or 10 mg/mL, XactEdit™ Cas9 Nuclease is formulated to perform highly efficient guide RNA (gRNA) targeted DNA digestion. Purified to >90% homogeneity, XactEdit™ Cas9 exhibits no non-specific nuclease activity. These properties, along with low endotoxin levels, make XactEdit™ Cas9 an ideal choice for a variety of applications. The XactEdit™ enzyme can be used for in vitro cleavage assays or introduced into cells by electroporation or transfection to target genomic sequences. Unlike vector or mRNA-based Cas9 systems, the XactEdit™ Cas9 RNP complex does not require transcription or translation and can act immediately after entering a cell.

Summary

  • Highly purified – Greater than 90% purity by Reverse Phase-HPLC and SDS-PAGE
  • Quality-controlled for high activity – lot testing by in vitro activity assay
  • No non-specific DNase or RNase activity
  • Low endotoxin levels – less than 0.01 EU/µg by LAL assay
  • Contains nuclear localization signal (NLS) for efficient targeting of the nucleus
  • Available at 1 mg/mL and 10 mg/mL concentrations
XactEdit cas9 Nuclease Kit

XactEdit™ Cas9 Nuclease with NLS
FIGURE 1. Overview of CRISPR/Cas9 Workflow to Evaluate gRNA Efficiency.

XactEdit™ Cas9 protein can be used for the evaluation of the effectiveness of a gRNA to perform in vitro cleavage. In situations where the efficiency and specificity of a gRNA are not known, multiple gRNA molecules can be analyzed using an in vitro digestion assay to identify the most effective gRNA and target site (Figure 1).

For cell-based assays, the Cas9 protein and gRNA are complexed and introduced into the cell by electroporation or lipid-mediated delivery. To evaluate off-target effects, a scrambled gRNA control is often run in parallel. Inside the cell, the Cas9:gRNA complex will mediate target-specific double-stranded DNA cleavage. The DNA break can be repaired by non-homologous end joining (NHEJ) to mediate a gene knockout or homologous recombination (HDR) to insert a specific DNA sequence or generate specific mutations. Changes to the genome can be detected by (1) single nucleotide polymorphism (SNP) assay to detect any site-specific insertion, deletion, or substitution, (2) amplification of the target sequence for sequencing, or (3) whole genome sequencing.

XactEdit™ Cas9 Nuclease with NLS Demonstrates High Levels of In Vitro Activity

In Vitro Activity Using Different amounts of XactEdit™ Cas9 Nuclease (NLS) and gRNA
FIGURE 2. In Vitro Activity Using Different amounts of XactEdit™ Cas9 Nuclease (NLS) and gRNA. To determine the most effective amount of XactEdit™ Cas9 Nuclease and gRNA to use, an in vitro activity assay was performed. In each lane, 100 ng of a 770 bp DNA fragment corresponding to the homeobox transcription factor Emx1 was incubated with indicated amount of XactEdit™ Cas9 protein and gRNA. Specific cleavage by the XactEdit™ Cas9: gRNA complex generates two fragments (283 bp and 487 bp). The data indicate that XactEdit™ Cas9 protein effectively and specifically cleaves the substrate at the target site using various amounts of input enzyme and gRNA. Molecular weight (MW) marker shown in lane 1 is 1 kb ladder.

XactEdit™ Cas9 Nuclease with NLS Demonstrates High and Consistent In Vivo Nuclease Activity

In Vivo Activity of XactEdit™ Cas9 Nuclease with NLS Compared to Other Commercially Available Ca9 (NLS) Nucleases
FIGURE 3. In Vivo Activity of XactEdit™ Cas9 Nuclease with NLS Compared to Other Commercially Available Cas9 (NLS) Nucleases. The in vivo nuclease activity of two different lots of XactEdit™ Cas9 nuclease were compared to that of Cas9 nuclease from supplier T and N. For each assay, Cas9:gRNA complexes were formed using 1 µg of Cas9 and 200 ng of in vitro transcribed guide RNA targeting the Emx1 gene. Complexes were introduced into HEK 293T cells by electroporation. Cas9 double strand break (DSB) frequency was examined by a mutation detection assay (panel A, left) and MiSeq™ analysis (panel B, right). In panel A, sequences created by DSB are shown as cleaved products in agarose gel electrophoresis indicating Cas9 induced DSB sites. Lanes 1 and 8 are molecular weight (MW) markers (Quick-Load® 2-Log DNA ladder from New England Biolabs). In panel B, quantitative analysis of MiSeq™ sequencing of the target site is shown as a percentage of cleavage of transfected cells. Data are presented as the mean with standard deviation of three replicates.

Highly Purified to Ensure Reliable, Consistent, and Specific Nuclease Activity

In Vivo Activity of XactEdit™ Cas9 Nuclease with NLS Compared to Other Commercially Available Ca9 (NLS) Nucleases
FIGURE 4. Reverse-phase HPLC analysis of XactEdit™ Cas9. 10 μg of XactEdit™ Cas9 was analyzed using reverse-phase HPLC. Zorbax 300SB-C3 column, mobile phase A = 0.1% TFA in water, mobile phase B = 0.1% TFA in acetonitrile, gradient = 5-100% B over 30 min at 1.0 mL/min, 40 °C, 214 nm detection.
Reverse-phase HPLC analysis of XactEdit™ Cas9
FIGURE 5. Reducing SDS-PAGE of XactEdit™ Cas9. XactEdit Cas9 was analyzed using reducing SDS-PAGE. 4-15% Bio-Rad TGX gel, Bio-Rad Precision Plus Protein Markers, SYPRO-Orange stain.
Lot to lot activity comparison of XactEdit™ Cas9
FIGURE 6. Lot to lot activity comparison of XactEdit™ Cas9. In vitro activity assay comparing the activity of two different lots of XactEdit™ Cas9 using three different guide RNAs (gRNA-1, gRNA-2 and gRNA-2) designed against a 5.5 kb target. gRNA-SC is a scrambled negative control RNA that does not recognize the target DNA. Presence or absence of the XactEdit™ Cas9 enzyme is represented by + and – respectively. Samples were analyzed by agarose gel electrophoresis to separate template DNA (5.5 kb, uncut) and digested products (variable sizes).

XactEdit™ Cas9 Nuclease with NLS

Products Catalog # Pack Size List Price
XactEdit™ Cas9 Nuclease (NLS) kit
Includes 10X XactEdit™ Digestion Buffer, Template for scrambled gRNA, Nuclease-free water
CE1000-50K 50 µg $ 149
XactEdit™ Cas9 Nuclease (NLS) 1 mg/mL CE1000-50 50 µg $ 139
XactEdit™ Cas9 Nuclease (NLS) 1 mg/mL
CE1000-250 5 x 50 µg $ 689
XactEdit™ Cas9 Nuclease (NLS) 10 mg/mL
CE1001-250 250 µg $ 689
XactEdit™ Cas9 Nuclease (NLS) 10 mg/mL
CE1001-1000 1000 µg $ 2,749

Place Your Order

Sign-in/Sign-up to your SGI-DNA account
Select "Enzymes and Cell Lines" and click " Enzymes"
Select your product
Check out

-or-
Inside North America Call: 855-4SGI-DNA (855-474-4362)
Outside North America Call: 858-288-4115
Email: customerservice@sgidna.com

If you have a promotional code that you want to be applied to your order, please e-mail us the order form at customerservice@sgidna.com