CRISPRcraft™ S.p. Cas9 Nuclease
Cas9 nuclease with NLS for targeted DNA cleavage
- Recombinant wild-type Streptococcus pyogenes Cas9 nuclease, with NLS
- Ideal for efficient RNP-based delivery of Cas9/gRNA complexes
- High concentration (10 mg/mL, 62 µM) compatible with multiple delivery methods
- Extensive quality control to ensure activity and purity
Free Sample Available
CRISPR-Cas9 gene editing is based on activity of Cas9 nuclease, an RNA-guided endonuclease (RGEN). Cas9 catalyzes sequence-specific cleavage of double-stranded DNA by forming a ribonucleoprotein (RNP) complex with a small (~97 nt) guide RNA that directs Cas9 to the desired locus. This precise DNA cleavage enables a variety of in vivo and in vitro applications including gene editing, CRISPR-mediated DNA detection, sequence enrichment and depletion.
CRISPRcraft™ S.p. Cas9 Nuclease is a purified recombinant wild-type Streptococcus pyogenes Cas9 nuclease produced in E. coli, containing a C-terminal 6xHis tag and a C-terminal NLS to allow efficient transport to the nucleus. It is provided at high concentration (10 mg/mL, 62 µM) to enable efficient RNP delivery and compatibility with multiple delivery methods including lipid-based transfection and electroporation (Figures 1 and 2).
Efficient In Vivo Delivery of CRISPRcraft™ S. p. Cas9 Nuclease
Figure 1: CRISPR RNP delivery.
HEK293T cells were transfected in triplicate with RNP containing CRISPRcraft™ S.p. Cas9 Nuclease and guide components from the CRISPRcraft™ S.p. Cas9 Nuclease Control Kit targeting HPRT, using either lipofection or electroporation. Transfection conditions: TransIT-X2® Dynamic Delivery System (Mirus), 50 nM gRNA and 25 nM Cas9; Lipofectamine® RNAiMAX (ThermoFisher Scientific), equimolar Cas9 and gRNA at 25 nM; Ingenio® Electroporation Solution (Mirus), 1.5 µM gRNA and 0.75 µM Cas9, plus Alt-R® Cas9 Electroporation Enhancer (IDT). At 48 hours post-transfection, cleavage efficiency at the HPRT locus was determined using the T7E1 mismatch detection assay and is expressed as % gene modification.
In Vivo Gene Editing
Figure 2: In vivo gene editing with different guide RNA sequences.
HEK293T cells were independently transfected in triplicate with RNP containing CRISPRcraft™ S.p. Cas9 Nuclease and three different guides, each targeting a different loci within human HPRT. Transfections were performed using TransIT-X2® Dynamic Delivery System (Mirus), 20 nM guide and 10 nM Cas9 (2:1 guide:Cas9 molar ratio). Cleavage efficiency at each HPRT locus was determined using the T7E1 mismatch detection assay, 48 hours post-transfection.
High quality of each lot of CRISPRcraft S.p. Cas9 Nuclease is guaranteed by ISO-13485-compliant manufacturing and quality systems, along with rigorous quality control assays (Table 1) to ensure enzyme purity and activity.
|Assay ||Acceptance Criteria |
|Purity ||≥ 95%, by SDS-PAGE |
|Endotoxin ||≤ 10 EU/mg, by LAL |
|Endonuclease Activity ||< 10% nicking as determined by agarose gel electrophoresis, 150 ng S.p. Cas9 per assay. |
|Exonuclease Activity ||No detectable DNA degradation as determined by agarose gel electrophoresis, 150 ng S.p. Cas9 per assay. |
|RNase Activity ||< LOD, by fluorescence-based assay, 15 µg S.p. Cas9 per assay (detection limit equivalent to 0.5 pg RNase A). |
|DNA Contamination ||< LOD, by agarose gel electrophoresis. |
|Activity Assay ||< 80% of linearized target DNA at 2 nM is digested with 20 nM S.p. Cas9 with equal molar ratio of guide RNA at 37°C for 10 min. |
Table 1. CRISPRcraft S.p. Cas9 Nuclease quality control specifications.
Take control of your CRISPR experiments with our CRISPRcraft™ S.p. Cas9 Nuclease Control Kit, containing positive control human HPRT guide RNA in vivo or in vitro CRISPR experiments, HPRT PCR primers to generate amplicons for mutation detection assays, HPRT substrate DNA and reaction buffer for in vitro cleavage assays. Click here to learn more or add the Control Kit to your order below.
CRISPRcraft S.p. Cas9 Nuclease is provided as a 12 µL (120 µg) or 40 µL (400 µg) size. Contact us for pricing on bulk enzyme orders.
The CRISPRcraft S.p. Cas9 Nuclease Control Kit contains Cas9 Control HPRT Guide crRNA and Cas9 Universal tracrRNA, Human HPRT Forward and Reverse PCR Primers, Control HPRT Substrate DNA and 10X RGEN Buffer. The Control Kit does not contain Cas9 enzyme.