Cas9 Protein (S. pyogenes), SpCas9, wildtype, with N- and C- terminal NLS

Description

Recombinant Cas9 Protein for CRISPR Applications: SpCas9

• Recombinant wt SpCas9 for forming RNP complexes with guide-RNA
• Highly purified and extremely active (blunt end ds DNA breaks)
• 2 NLS sites for delivery into the nucleus and high efficiency genome editing

CasRNPs are an improvement on plasmid transfections because the Cas protein is cleared from the system within 24 hours, decreasing off-target effects. The method also saves time, as the active complex is what’s being delivered straight to the nucleus of the cell.  Editing efficiency is correlated with Cas protein purity, so it’s crucial that high quality Cas9 such as the ultra-pure TriaAltus Bioscience Cas9 protein is used.

TriAltus’ highly purified recombinant Streptococcus pyogenes Cas9 (wt) protein (>99% purity) with two (N-terminal and C-terminal) nuclear localization signals (NLS) is used for genome editing with CRISPR technology and CRISPR-mediated in vitro applications.  The enzyme was expressed in E. coli with TriAltus´ CL7 tag, which was removed with PreScission protease during the purification process. SpCas9 protein combines with guide RNA (gRNA) to form a stable ribonucleoprotein (RNP) complex that enables the inactive nuclease to be guided to the specific double stranded DNA (dsDNA) site. Once guided to the specific cleavage site, SpCas9 becomes activated to specifically and precisely cleave both DNA strands creating blunt ends.

Why use ultra-pure Cas9?

In the past several years, CRISPR has emerged as a powerful system for targeted gene modification. The system requires only two primary components: a guide RNA (gRNA or sgRNA) sequence and a Cas9 enzyme. Cas9 is essential for initiating a double-stranded break in the gene sequence at the site to which the gRNA guides it. In order to achieve high-fidelity results, the Cas9 protein must be pure and active. Purity and activity are largely determined by the method of purification that is used to isolate the Cas9 protein. Incumbent methods require multiple purification steps that can include combinations of metal affinity enrichment, cation exchange chromatography, size exclusion chromatography, and tag-based affinity chromatography. Depending on multiple chromatography steps for purification is not only time consuming, but also results in product losses at each phase and therefore, a lower final yield. Trialtus´ CL7/lm7 system enables a highly efficient one step purification of extremely pure and active Cas9. Recently, researchers at Hubei University (Wuhan, China) used Trialtus´ CL7/Im7 system to purify even Cas9 ribonucleoproteins (RNPs) in one step (Qiao, et al. Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins. Commun Biol 2, 161 (2019).

SPECIFICATIONS

PURITY | ~99% (SDS-PAGE gel analysis)

ENZYME SOURCE | Streptococcus pyogenes Cas9 is expressed in E. coli with two (N-terminal and C-terminal) nuclear localization signals (NLS), and is purified with TriAltus Biosciences´ CL7 tag technology. The protein is provided tag-free.

ENDOTOXIN | <0.3 EU/ug by rFC method

QUANTITY | 100 ug

STORAGE/SHIPPING CONCENTRATION | 5 mg/mL

STORAGE BUFFER | 10 mM Tris-Cl pH 8.0, 250 mM NaCl, 50% glycerol

RECOMMENDED STORAGE CONDITIONS | -80°C

EXPIRATION | 6 months from receipt when stored as directed. Avoid repeated freeze/thaw cycles.