A detailed protocol for expression, purification, and activity determination of recombinant SaCas9

An Efficient Expression and Purification Protocol for SpCas9 Nuclease and Evaluation of Different Delivery Methods of Ribonucleoprotein

“…In this study, we introduce a method for the expression and purification of the SpCas9 nuclease as a fusion protein with a SUMO-tag. Despite the existence of several variations of SpCas9 isolation strategy in the literature [13][14][15][16][17]23], the vast number of studies utilizing SpCas9 requires the development of new reproducible protocols for obtaining Simultaneously, we assessed the protein lifetime in cells after transfection with plasmid or RNP complex using Western blotting at 1, 3, 6 and 9 days after transfection (Figure 3c). As expected for transfection with a plasmid encoding Cas9, we detected comparable protein levels on days 1, 3, and 6 in cell culture.…”

“…However, due to SpCas9's size and cell toxicity [12], obtaining this protein in sufficient quantities presents significant challenges. Numerous production methods have been described, employing different expression strains, induction conditions, and isolation and purification techniques [13][14][15][16][17]. These methods are primarily based on metal-affinity chromatography, followed by gel filtration or ion-exchange column purification.…”

An efficient and cost-effective purification protocol for Staphylococcus aureus Cas9 nuclease

“…Purified SaCas9 can be directly used for in vitro applications. The new methodology is superior to the majority of conventional approaches, which rely on expensive infrastructure ( E.g., French press, high frequency sonicator, or fast protein liquid chromatography), 5 , 6 , 7 , 8 and thereby rendering SaCas9 production more cost-efficient. The average cost for the production of 10 mg of SaCas9 proteins is 86.36 USD ( Table 1 ), significantly lesser than several commercial sources, without compromising the quality of the enzyme itself.…”

“… 3 , 4 However, acquiring this protein remains a challenge for many laboratories that are not adequately equipped for protein purification. 5 , 6 , 7 , 8 , 9 For instance, fast protein liquid chromatography was used for purifying Cas9 proteins in several studies and the equipment requires a hefty initial investment. 6 , 7 , 8 Here, we report an advancement of the purification methods for SaCas9 from bacterial cells.…”

“… 5 , 6 , 7 , 8 , 9 For instance, fast protein liquid chromatography was used for purifying Cas9 proteins in several studies and the equipment requires a hefty initial investment. 6 , 7 , 8 Here, we report an advancement of the purification methods for SaCas9 from bacterial cells. A major advantage of this methodology is to achieve over 90% purity, at large batch sizes (concentrations at 1 mg/L) within a day.…”