Abstract
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.