Purification of Cas9-RNA complexes by ultrafiltration

The discovery of CRISPR-Cas9 has revolutionized molecular biology, greatly accelerating the introduction of genetic modifications into organisms and facilitating the development of novel therapeutics and diagnostics. For many applications, guide RNA and Cas9 protein are expressed, combined, and purified to produce a ribonucleic enzyme complex that is then added into a diagnostic device or delivered into cells. The objective of this work was to develop an ultrafiltration process for the selective purification of Cas9 ribonucleoprotein by removal of excess guide RNA. A His-tagged Streptococcus pyogenes Cas9 protein was produced in Escherichia coli, purified by metal affinity chromatography, and complexed with a 40 kDa (124 nucleotide) single guide RNA. Ultrafiltration experiments were first performed on solutions containing either guide RNA or Cas9 protein to identify the effect of filtration conditions and membrane pore size on the selectivity. Shear-induced aggregation of the Cas9 led to significant fouling under some conditions. A diafiltration process was then developed using a Biomax® 300 kDa polyethersulfone membrane to selectively remove excess guide RNA from a solution containing Cas9-bound guide RNA and free guide RNA. These results demonstrate the potential of using ultrafiltration for the removal of excess RNA during the production of functional ribonucleoprotein complexes.