Enhanced production of functional CRISPR-AsCas12a protein in Escherichia coli

Abstract

The CRISPR-Cas12a system is a groundbreaking tool widely used for genome editing and diagnostics in biotechnology and biomedicine research laboratories. Despite its growing application, studies optimizing Cas12a protein production at the laboratory scale using straightforward protocols remains scarce. This study aimed to enhance the lab-scale recombinant production of Acidaminococcus sp Cas12a protein (AsCas12a) in E. coli. Through targeted adjustments of simple parameters, AsCas12a production was significantly increased. The optimized conditions included the use of E. coli BL21(DE3), TB medium supplemented with 1 % glucose, induction with 0.3 mM IPTG for at least 6–9 h, and incubation at 30 °C. Notably, these conditions differ from conventional protocols typically used for Cas12a and related proteins, such as Streptococcus pyogenes Cas9. Upon combining all optimized parameters, AsCas12a production increased approximately 3-fold, from 0.95 mg/mL of bacterial lysate under non-optimized conditions to 3.73 mg/mL under optimized ones. After chromatographic purification, the final protein yield rose approximately 4.5-fold, from 5.2 to 23.4 mg/L of culture volume, without compromising functional activity. The purified AsCas12a retained full activity for programmable in vitro DNA cis-cleavage and collateral trans-cleavage, which was successfully applied to detect the N gene of SARS-CoV-2. This optimized method provide an efficient and high-yield approach for producing functional AsCas12a protein using accessible materials and conditions available to many research laboratories worldwide.