Recombinant Cas9 protein production in an endotoxin-free system and evaluation with editing the BCL11A gene in human cells

Abstract

Many therapeutic proteins are expressed in Escherichia coli bacteria for the low cost and high yield obtained. However, these gram-negative bacteria also generate undesirable endotoxin byproducts such as lipopolysaccharides (LPS). These endotoxins can induce a human immune response and cause severe inflammation. To mitigate this problem, we have employed the ClearColi BL21 (DE3) endotoxin-free cells as an expression host for Cas9 protein production. Cas9 is an endonuclease enzyme that plays a key role in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (CRISPR/Cas9) genome editing technique. This technology is very promising for use in diagnostics as well as treatment of diseases, especially for genetic diseases such as thalassemia. The potential uses for this technology thus generate a considerable interest for Cas9 utilization as a therapeutic protein in clinical treatment. Therefore, special care in protein production should be a major concern. Accordingly, we expressed the Cas9 protein in endotoxin-free bacterial cells achieving 99% purity with activity comparable to commercially available Cas9. Our protocol therefore yields a cost-effective product suitable for invitro experiments with stem cells.

Keywords: CRISPR/Cas9; Endotoxin; Lipopolysaccharide; Ribonucleoprotein; Therapeutic protein.