Highly purified recombinant Streptococcus pyogenes Cas9 (wt) protein (>99% purity) with two (N-terminal and C-terminal) nuclear localization signals (NLS). The enzyme was expressed in E. coli with TriAltus´ CL7 tag, which was removed with PreScission protease during the purification process. TriAltus’ highly pure Cas9 protein is designed for genome editing with CRISPR technology. The Cas9 protein combines with guide RNA (gRNA) to form a stable ribonucleoprotein (RNP) complex that enables efficient and precise 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 Cas9 protein is used.
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).