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
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.
|