Background Conventional methods applied to develop recombinant CHO (rCHO) cell line as a predominant host for mammalian protein expression are limited to random integration approaches, which can prolong the process of getting the desired clones for months. CRISPR/Cas9 could be an alternative by mediating site-specific integration into transcriptionally active hot spots, promoting homogenous clones, and shortening the clonal selection process. However, applying this approach for the rCHO cell line development depends on an acceptable integration rate and robust sites for the sustained expression. Methods and results In this study, we aimed at improving the rate of GFP reporter integration to the Chromosome 3 (Chr3) pseudo-attP site of the CHO-K1 genome via two strategies; these include the PCR-based donor linearization and increasing local concentration of donor in the vicinity of DSB site by applying the monomeric streptavidin (mSA)-biotin tethering approach. According to the results, compared to the conventional CRISPR-mediated targeting, donor linearization and tethering methods exhibited 1.6- and 2.4-fold improvement in knock-in efficiency; among on-target clones, 84% and 73% were determined to be single copy by the quantitative PCR, respectively. Finally, to evaluate the expression level of the targeted integration, the expression cassette of hrsACE2 as a secretory protein was targeted to the Chr3 pseudo-attP site by applying the established tethering method. The generated cell pool reached 2-fold productivity, as compared to the random integration cell line. Conclusion Our study suggested reliable strategies for enhancing the CRISPR-mediated integration, introducing Chr3 pseudo-attP site as a potential candidate for the sustained transgene expression, which might be applied to promote the rCHO cell line development. Supplementary information The online version contains supplementary material available at 10.1007/s11033-023-08529-8.

CRISPR-mediated integration could be used to develop the recombinant CHO (rCHO) cells by knock-in into the hotspot loci. However, low HDR efficiency besides the complex donor design is the main barrier for achieving so. The recently introduced MMEJ-mediated CRISPR system (CRIS-PITCh) uses a donor with short homology arms, being linearized in the cells via two sgRNAs. In this paper, a new approach to improve CRIS-PITCh knock-in efficiency by employing small molecules was investigated. Two small molecules, B02, a Rad51 inhibitor, and Nocodazole, a G2/M cell cycle synchronizer, were used to target the S100A hotspot site using a bxb1 recombinase comprised landing pad in CHO-K1 cells. Following transfection, the CHO-K1 cells were treated with the optimum concentration of one or combination of small molecules, being determined by the cell viability or flow cytometric cell cycle assay. Stable cell lines were generated and the single-cell clones were achieved by the clonal selection procedure. The finding showed that B02 improved the PITCh-mediated integration approximately twofold. In the case of Nocodazole treatment, the improvement was even more significant, up to 2.4-fold. However, the combinatorial effects of both molecules were not substantial. Moreover, according to the copy number and out-out PCR analyses, 5 and 6 of 20 clonal cells exhibited mono-allelic integration in Nocodazole and B02 groups, respectively. The results of the present study as the first attempt to enhance the CHO platform generation by exploiting two small molecules in the CRIS-PITCh system could be used in future researches to establish rCHO clones.