High Throughput, Efficacious Gene Editing & Genome Surveillance in Chinese Hamster Ovary Cells

Purpose: Chinese hamster ovary (CHO) cells are a common tool utilized in bioproduction and directed genome engineering of CHO cells is of great interest to enhance recombinant cell lines. Until recently, this focus has been challenged by a lack of efficacious, high throughput, and low-cost gene editing modalities and screening methods. In this work, we demonstrate an improved method for gene editing in CHO cells using CRISPR RNPs and characterize the endpoints of Cas9 and ZFN mediated genetic engineering. Methods: Amplicons were first assayed for quality using an Invitrogen Quant-iT dsDNA (Thermo, Cat Q33120) assay and gel electrophoresis to determine DNA concentration and DNA quality. Samples were then used for Sanger sequence based decomposition or processed downstream using NGS. For NGS, libraries were generated using the Illumina TruSeq Nano DNA kit (Illumina, Cat: 20015964). The libraries were sequenced using the Illumina MiSeq platform, with read length of 2x150bp. 4.5Gb of sequencing data was generated per DNA sample. Results: There was a tendency for decomposition to underestimate editing as compared to NGS as the indel size became larger. Despite these results, sequence decomposition was able to reproduce next-generation data in the context of gene editing efficiency across a short amplicon. Conclusions: We took advantage of the differential Indel spectrums induced by both ZFN and CRISPR to validate metrics for assess gene editing and show how deconvolution-based methods, can be utilized in the context of cell line development. Amplicons from ZFN or CRISPR modified cell pools were analyzed 48-72 hours after transfection. The cells were lysed, amplified by PCR, and the amplicons were sequenced. For clones, cells were FACs sorted and allowed to grow for approximately 14 days. The clones were then analyzed using the method described above.