Optimizing Heterologous Production of CRISPR-AsCas12a Protein in Escherichia coli

The CRISPR-Cas12a system is a groundbreaking tool that has seen an ample use for genome editing and diagnostics in biotechnology and biomedicine research labs. Despite its increasing use, there is a lack of studies on optimizing Cas12a protein production at lab-scale using straightforward protocols. This study aimed on enhancing the lab-scale recombinant production of Acidaminococcus sp Cas12a protein (AsCas12a) in E. coli . Through careful adjustments of simple parameters, the production of AsCas12a was remarkably increased. Optimized conditions involved using the BL21(DE3) strain, TB medium with 1% glucose, induction with 0.3 mM IPTG for at least 6–9 h and incubation at 30°C. Notably, these conditions deviate from conventional production protocols for Cas12a and related proteins such as Cas9 from Streptococcus pyogenes . Upon combination of all optimized conditions bacterial production of AsCas12a improved ~ 3 times, passing from 0.95 mg / mL of bacterial lysate volume, for non-optimized conditions, to 3.73 mg/mL in the optimal ones. The production yield of AsCas12a protein, after chromatographical purification increased ~ 4.5 times, from 5.2 to 23.4 mg/L (culture volume) without compromising its functionality at all. The purified AsCas12a protein retained full activity for programmable in vitro DNA cis -cleavage and for collateral trans -activity, which was used to detect the N gene from SARS-CoV-2. This optimized method offers an efficient and high-yield AsCas12a protein production using materials and conditions that are accessible to many research labs around the world.