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Baer, Alexandra

Beatriz Bulte Ocaña, Dubhe

Grav, Lise M.

Ravn, Peter

English

Engineering Conferences International

 Poster Number 52 CELL LINES DEVELOPMENT THROUGH TARGETED INTEGRATION INVOLVING A CRISPR-MAD7-BASED SYSTEM IN CHO-DG44  Dubhe Beatriz Bulté Ocaña, Technical University of Denmark (DTU) dbboc@dtu.dk Lise M. Grav, Technical University of Denmark (DTU) Alexandra Baer, Bioneer Peter Ravn, Bioneer   Key Words: Cell line development, CHO-DG44, Targeted Integration, CRISPR, Mad7 nuclease.  CHO cells are the primary expression platform for therapeutic protein production. Most processes for cell line development involve random or semi-random transgene integration which lead to high phenotypic heterogeneity that can cause loss of expression. However, advances in genome-editing technologies, and knowledge-based selection of integration sites have made it possible to use targeted integration (TI) methods as a suitable way to develop cell lines with predictable and consistent transgene expression. Previously, we have reported how our TI strategy, based on a combination of CRISPR-Cas and Recombinant Mediate Cassette Exchange (RMCE) tools, has shown an efficient and stable transgene integration into different safe harbor sites identified for CHO-S cells (1). On the other hand, considering the extensive utilization of CHO-DG44 by the biopharmaceutical industry, and the advantages for commercial use of Mad7 nuclease, we aim to set the more effective conditions using a CRISPR-Mad7 system to generate a CHO-DG44 master cell lines (MCL) for targeted expression of recombinant proteins. For this purpose, the conservation of “T9” and “A” safe harbor sites sequences in CHO-DG44 were confirmed. We further assessed the Mad7 nuclease and the Indel efficiency of designed gRNAs. The proficient performance of Mad7 was verified by successfully generated MCLs with site-specifically integrated lading-pads (LPs). Co-transfection with three vectors involved for LP integration, was optimized for CHO-DG44 to reach an efficiency around 65% which was lower than CHO-S (90%), but it was shown to be suitable for the CRISPR mechanism. Interestingly, for recombinant CHO-DG44 that have integrated a first LP (rCHO-DG44-MCL) restoring dhfr, the transfection efficiency improved up to levels higher than 90% for a second LP integration. We also showed that antibiotics is a suitable choice for the selection of LPs-rCHO-DG44 cells, considering that the antibiotic resistance gene can be removed after the RMCE stage, and dhfr can be included in the donor cassette, to avoid the auxotrophic media supplementation during the production process. Overall, this study demonstrates an optimal method for targeted integration using CRISPR/Mad7 in CHO-DG44 and valuable insights on how to implement a CRISPR-Mad7-based system for a faster and more efficient cell line development through a TI approach.      (1) Nuša Pristovšek, Saranya Nallapareddy, Lise Marie Grav, Hooman Hefzi, Nathan E. Lewis, Peter Rugbjerg, Henning Gram Hansen, Gyun Min Lee, Mikael Rørdam Andersen, and Helene Faustrup Kildegaard. 2019. Systematic Evaluation of Site-Specific Recombinant Gene Expression for Programmable Mammalian Cell Engineering. ACS Synthetic Biology 8(4), 758-774. DOI: 10.1021/acssynbio.8b00453            

Cell lines development through targeted integration involving a CRISPR-Mad7-based system in CHO-DG44

https://dc.engconfintl.org/cgi/viewcontent.cgi?filename=0&article=1099&context=ccexviii&type=additional

Cell lines development through targeted integration involving a CRISPR-Mad7-based system in CHO-DG44

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Engineering Conferences International