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

Teng, Allen C. T.; Tavassoli, Marjan; Shrestha, Suja; Marks, Ray; McFadden, Meghan J.; Evagelou, Sonia L.; Lindsay, Kyle; Vandenbelt, Ava; Ivakine, Evgueni A.; Cohn, Ronald D.; Santerre, J. Paul; Gramolini, Anthony O.

doi:10.1016/j.xpro.2022.101933

Cited by 3 publications

(5 citation statements)

References 10 publications

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“…The cells were collected after 6 h of induction, as described previously. Next, four different induction temperatures (18,25,30, and 37 • C) were evaluated. The cell lysate corresponding to each condition was analyzed by SDS-PAGE and the protein expression was found to be highest when induced at 30 • C. The results are presented in Figure 5C.…”

Section: Anticipated Resultsmentioning

confidence: 99%

“…Despite the suitability of E. coli for heterologous expression, challenges such as the large size of SpCas9 and the presence of rare codons may impact overall yield. Existing protocols lack standardization and vary in bacterial strains, culture conditions, and expression vectors [ 17 , 18 ]. The optimization process involves evaluating competent cell strains, IPTG concentrations, post-induction times, and temperatures to ensure scalability for industrial production [ 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

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Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing

Agrawal,

Padmaswari,

Stokes

et al. 2024

Biomedicines

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The CRISPR-Cas9 system is a revolutionary tool in genetic engineering, offering unprecedented precision and efficiency in genome editing. Cas9, an enzyme derived from bacteria, is guided by RNA to edit DNA sequences within cells precisely. However, while CRISPR-Cas9 presents notable benefits and encouraging outcomes as a molecular tool and a potential therapeutic agent, the process of producing and purifying recombinant Cas9 protein remains a formidable hurdle. In this study, we systematically investigated the expression of recombinant SpCas9-His in four distinct Escherichia coli (E. coli) strains (Rosetta2, BL21(DE3), BL21(DE3)-pLysS, and BL21(DE3)-Star). Through optimization of culture conditions, including temperature and post-induction time, the BL21(DE3)-pLysS strain demonstrated efficient SpCas9 protein expression. This study also presents a detailed protocol for the purification of recombinant SpCas9, along with detailed troubleshooting tips. Results indicate successful SpCas9 protein expression using E. coli BL21(DE3)-pLysS at 0.5 mM IPTG concentration. Furthermore, the findings suggest potential avenues for further enhancements, paving the way for large-scale Cas9 production. This research contributes valuable insights into optimizing E. coli strains and culture conditions for enhanced Cas9 expression, offering a step forward in the development of efficient genome editing tools and therapeutic proteins.

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Section: Anticipated Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing

Agrawal,

Padmaswari,

Stokes

et al. 2024

Biomedicines

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show abstract

“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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 functionally active protein. A key advantage and novelty of our approach is its high productivity and simplicity, which allowed us to obtain an extremely large amount of SpCas9 in bacterial cells.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Evmenov,

Pustogarov,

Panteleev

et al. 2024

IJMS

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The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a revolutionary tool for precise genome editing across various cell types. Ribonucleoproteins (RNPs), encompassing the Cas9 protein and guide RNA (gRNA), have emerged as a promising technique due to their increased specificity and reduced off-target effects. This method eliminates the need for plasmid DNA introduction, thereby preventing potential integration of foreign DNA into the target cell genome. Given the requirement for large quantities of highly purified protein in various Cas9 studies, we present an efficient and simple method for the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein. This method leverages the Small Ubiquitin Like Modifier(SUMO) tag system, which includes metal-affinity chromatography followed by anion-exchange chromatography purification. Furthermore, we compare two methods of CRISPR-Cas9 system delivery into cells: transfection with plasmid DNA encoding the CRISPR-Cas9 system and RNP transfection with the Cas9-gRNA complex. We estimate the efficiency of genomic editing and protein lifespan post-transfection. Intriguingly, we found that RNP treatment of cells, even in the absence of a transfection system, is a relatively efficient method for RNP delivery into cell culture. This discovery is particularly promising as it can significantly reduce cytotoxicity, which is crucial for certain cell cultures such as induced pluripotent stem cells (iPSCs).

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A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications

Pandey,

Yadav,

Shah

et al. 2024

Protein Expression and Purification

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

Cited by 3 publications

References 10 publications

Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing

Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing

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

A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications

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