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

Abstract

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).

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

“…As per existing literature, optimized E. coli strains, such as Rosetta2, have been commonly chosen for SpCas9-His protein expression; however, the reported studies vary significantly in expression conditions, displaying concerns about productivity and scalability. Previous research employed complex culture media, low induction temperatures, and extended induction times, often associated with challenges and low SpCas9-His protein expression levels [ 21 , 27 , 28 , 29 ]. Moreover, these studies lacked stringent control of bacterial growth in bioreactors.…”

“…In this study, SpCas9-His protein expression was initially assessed using the temperature and IPTG concentration conditions (0.5 mM IPTG at 18 °C overnight) from the existing literature [ 21 ]. Consequently, we conducted small-scale expression experiments (in shake flasks with 50 mL of LB media) of SpCas9-His protein with various bacterial strains, including BL21(DE3)-pLysS, BL21(DE3)-Star, Rosetta2, and BL21(DE3), and found that BL21(DE3)-pLysS exhibited superior expression compared to others.…”

“…The optimization process involves evaluating competent cell strains, IPTG concentrations, post-induction times, and temperatures to ensure scalability for industrial production [ 19 , 20 ]. The significance of these efforts lies in bridging the gap between exploratory protein purification and large-scale industrial processes [ 21 , 22 ].…”

Effect of the Phosphoryl Guanidine Modification in Chimeric DNA–RNA crRNAs on the Activity of the CRISPR-Cas9 System In Vitro

“…Delivery of the CRISPR/Cas9 system into cells is a critical factor in gene editing. The RNP delivery method is the most common, simple, and effective approach to cell editing. − Using RNP minimizes immunological and toxic side effects, reduces off-target activity, , and achieves high editing efficiency . Additionally, RNP editing occurs rapidly, which prevents unintended genomic changes and ensures the method’s safety .…”