Journal article
Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing
Department of Biotechnology and Biomedicine, Technical University of Denmark1
Yeast Natural Products, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
Technical University of Denmark3
National Biologics Facility, Section for Protein Science and Biotherapeutics, Department of Biotechnology and Biomedicine, Technical University of Denmark4
Biotherapeutic Glycoengineering and Immunology, Section for Protein Science and Biotherapeutics, Department of Biotechnology and Biomedicine, Technical University of Denmark5
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark6
Section for Protein Science and Biotherapeutics, Department of Biotechnology and Biomedicine, Technical University of Denmark7
Mammalian Cell Line Engineering, Section for Protein Science and Biotherapeutics, Department of Biotechnology and Biomedicine, Technical University of Denmark8
Quantitative Modeling of Cell Metabolism, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark9
Department of Health Technology, Technical University of Denmark10
Experimental & Translational Immunology, Department of Health Technology, Technical University of Denmark11
T-Cells and Cancer, Experimental & Translational Immunology, Department of Health Technology, Technical University of Denmark12
Bacterial Signal Transduction, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark13
...and 3 moreThe production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese Hamster Ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements.
Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms.
In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data.
In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.
| Language: | English |
|---|---|
| Year: | 2023 |
| Pages: | 1478-1491 |
| ISSN: | 10970290 and 00063592 |
| Types: | Journal article |
| DOI: | 10.1002/bit.28367 |
| ORCIDs: | Rojek, Johan Blatt , Ocaña, Dubhe Beatriz Bulté , Pedersen, Lasse Ebdrup , Basavaraju, Yogesh , Schoffelen, Sanne , Grav, Lise Marie and Goletz, Steffen |
