Scalable Expression of LbCas12a Proteins in E. coli

CRISPR Cas proteins such as Cas12 and Cas9 proteins offer great opportunities for a wide range of sensing applications but at a high cost. When obtaining proteins for research commercially, it can cost the lab up to $1,000 for only 0.5 mg of LbCas12a. The purpose of my project over the summer was to determine if we could produce needed proteins via a low-cost bacterial expression method utilizing the equipment and materials available in our lab. If successful, it would also allow for the purchase of plasmids that would produce other Cas proteins that are not commercially available, giving our research lab a wider range of CRISPR protein options. The overall process involved 1) plating the plasmid containing LbCas12a from the agar stab, 2) purifying it via Qiagen Miniprep, 3) transforming the plasmid into E. Coli, and 4) cell growth and protein expression via fermentation. After fermentation, the cells were pelleted and lysed. Half of the cells were lysed via freeze/thaw and sonication methods. The other half of the cells were lysed by chemical lysis to compare the efficiency of each method. LbCas12a was purified and multiple BCA Assays were completed to determine yield. We had roughly 2 mg of protein after purification from a one-liter fermentation. Next steps include characterizing the efficiency of the protein through CRISPR diagnostic assays as well as determining protein purity with SDS-PAGE. An additional fermentation will also be conducted to enlarge the amount of culture and protein we can use.