CRISPR technologies have created exciting opportunities for gene editing and novel biotherapeutics, using an engineered single guide RNA (sgRNA) that binds to specific DNA sites enabling modification / regulation of targeted genes. The development of CRISPR-based therapeutics requires robust methods for downstream processing, including the purification ribonucleoprotein complex from residual free RNA. We present the first study on the potential use of ultrafiltration membranes to separate Cas9/sgRNA complex from free unbound single guide RNA. The Cas9 protein was produced in E. coli NiCo21 (DE3) cells transformed with pET-28a-Cas9-Cys plasmid containing the S. pyogenes Cas9 with a 6xHis tag. Cells were grown in a 5L bioreactor, lysed, and clarified, with the lysate purified by Nickel Affinity Chromatography using a 5 mL HisPure Ni-NTA column. A 48 kDa sgRNA was synthesized from a DNA template consisting of the T7 promoter sequence, a guanosine nucleoside to promote efficient initiation of transcription, and the appropriate DNA sequence. The DNA was prepared from a gBlock fragment and amplified by PCR. T7 RNA polymerase was used for in vitro RNA synthesis using the T7 HiScribe Kit. Synthesized RNA was purified using phenol:chloroform extraction and then coupled to the Cas9 protein. We performed stirred cell filtration experiments with the individual Cas9 and sgRNA to identify appropriate conditions for separation. Key variables were the membrane molecular weight cutoff (50 – 300 kDa), the membrane chemistries (PES and RC), and the filtration velocity. We separate the Cas9/sgRNA complex from free sgRNA via diafiltration, with samples analyzed by gel electrophoresis using SYBR Gold and SYPRO Ruby dyes for visualization of the RNA and Cas9, respectively. Preliminary data showed effective removal of the sgRNA using a Biomax 100 kD membrane at filtrate flux around 30 µm/s (100 LMH). These results demonstrate the potential of using ultrafiltration for the purification of ribonucleoproteins from free unbound RNA, providing a low-cost scalable system suitable for large-scale production of these new biotherapeutics.

Purification of Cas9/sgRNA complexes from residual sgRNA by ultrafiltration

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.