21/08/1402
Targeting DNA repair pathways with B02 and Nocodazole small molecules to improve CRIS-PITCh mediated cassette integration in CHO-K1 cells
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.