Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.

Therapeutic genome editing: prospects and challenges

Chinese hamster ovary (CHO) cells are utilized as primary mammalian expression host cells for the production of biopharmaceuticals. Recombinant CHO cell line development (CLD) has been a crucial step for therapeutic protein production platforms; however, this step remains time-consuming and costly. With the emergence of multiomics data sets of CHO cells and genome editing technology such as CRISPR/Cas9, site-specific integration-based cell line development, and engineering have been successfully implemented in CHO cells for predictable transgene expression and expediting the process of CLD. This review describes the trends in CHO CLD from random to targeted approaches. And we cover the major obstacles faced in rational CHO CLD and the potential strategies employed to overcome its limitations. Finally, we conclude by discussing future directions and challenges for next-generation CHO cell factories.

CHO Cell Line Development and Engineering via Site-specific Integration: Challenges and Opportunities

The discovery of clustered regularly interspaced palindromic repeats (CRISPR), a defense system against viruses found in bacteria, launched a new era in gene targeting. The key feature of this technique is the guiding of the endonuclease Cas9 by single guide RNAs (sgRNA) to specific sequences, where a DNA lesion is introduced to trigger DNA repair. The CRISPR/Cas9 system may be extremely relevant for gene therapy, but the technique needs improvement to become a safe and fully effective tool. The Cas9-induced double-strand break (DSB) is repaired by one of two pathways, the error-prone Non-homologous end joining (NHEJ) or the high-fidelity Homology Direct Repair (HDR). Shifting the repair of the DSB to HDR is challenging, given the efficiency of NHEJ. Here we describe an engineered protein approach to increase knock-in efficiency by promoting the relative increase in Cas9 activity in G2, the phase of the cell cycle where HDR is more active. Cas9 was fused to the degradation domain of proteins known to be degraded in G1. The activity of two chimeric proteins, Geminin-Cas9 and CyclinB2-Cas9, is demonstrated, as well as their cell-cycle-dependent degradation. The chimeras shifted the repair of the DSBs to the HDR repair pathway compared to the commonly used Cas9. The application of cell cycle specific degradation tags could pave the way for more efficient and secure gene editing applications of the CRISPR/Cas9 system.

/pdf/a-cyclinb2-cas9-fusion-promotes-the-homology-directed-repair-kwsi3w1uk6.pdf

A CyclinB2-Cas9 fusion promotes the homology-directed repair of double-strand breaks

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A CyclinB2-Cas9 fusion promotes the homology-directed repair of double-strand breaks