High‐efficiency and multilocus targeted integration in CHO cells using CRISPR‐mediated donor nicking and DNA repair inhibitors

Abstract

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all‐in‐one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site‐specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double‐cut and double‐nick donor formats significantly improved targeting accuracy by 2.3–8.3‐fold and 19–22‐fold, respectively, compared to standard circular donors. Notably, Cas9‐mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low‐fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR‐based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.

Advanced cell technologies: Making protein, cell, and gene therapies a reality

“…This method was able to identify integration sites, the composition of the integrated sequence, and the DNA methylation status in CHO cells in a single sequencing run. Building up on CRISPR/Cas9 technology for mammalian cells, Hamaker and Lee (2023) developed an all‐in‐one reporter system to quantify gene disruption and site‐specific integration (SSI) in CHO cells. Using this system, it was possible to identify specific molecules (inhibitors of DNA repair pathways) that enhance SSI efficiency and thus accelerate cell engineering.…”