• Investigating the effect of nocodazol small molecule on CRIS-PITCH efficiency for targeted integration of transgene Cassette in CHO cell
  • Behnaz rahmani,1 Mohammad Hassan Kheirandish,2 Fahime shamsi,3 Samaneh Ghanbari Mehrandooei2,4 Fateme davami,5,*
    1. Medical Biotechnology Department, Semnan University of Medical Sciences, Semnan, Iran
    2. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
    3. Medical Biotechnology Department, Semnan University of Medical Sciences, Semnan, Iran
    4. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
    5. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran


  • Introduction: CRISPR/Cas9 have been recently introduced as an alternative tool in CHO cell line development as a dominant expression host to integration of the transgene into the predetermined site of the genome via homologous recombination (HR) that promotes stable and high transgene expression. In this technology, donor plasmid harbors the transgene flanked by long homology arms that made vector designing complex and costly. Recently microhomology-mediated end joining (MMEJ)-based method termed Cris-Pitch (Precise Integration into Target Chromosome) have been used donor with in vivo linearized short homologous sequence (5–25 bp) to site specific integration. However this method still displays low targeted efficiency. To address this challenge, we combined Cris-Pitch system with nocodazole as a small molecule. We found that nocodazole-arrested mitotic cells use MMEJ to fix DSB damage. Indeed we further improved Cris-Pitch mediated knock-in efficiency using nocodazole to target the transgene to the transcriptionally active site of CHO-K1 cell line.
  • Methods: The CHO-K1 cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and 10% CO2 at 37°C incubator. The puromycin sensitivity was also determined. The day before transfection, approximately 65×103 cells/mL were cultured in 24-well cell culture plates. Cells in each well were co-transfected with 500 ng of Donor plasmid and 500 ng of all-in-one plasmid mixed with 3 µL of Lipofectamine 3000 and 2 µL p3000, in 50 µL of DMEM/F12. 31 hours after transfection cell culture medium was changed and media containing 40 ng/ml of nocodazole was added to the cells. (Cells in control group just received fresh medium without small molecule). CHO-K1 cells were incubated with nocodazole for 15 h, after 15 hour medium was changed with fresh medium. Cell synchronization have been evaluated by Flow cytometry. 72 hours after transfection, the cells were seeded in a 6-well plate and were selected with 3 µg/mL of puromycin for approximately 12 days to obtain antibiotic resistant cell pool. Cell pool with the desired genome changes have been confirmed with 5′/3′ junction PCR, limiting dilution was done into the 96-well plates at a density of 1 cell/well for the isolation of individual clones. About 10 days after seeding into 96 well, the single colonies in some wells had grown enough to transfer from 96 well to 24 well plates. Each recovered clone was examined by 5′/3′ junction PCR to evaluate the rate of knock-in efficiency.
  • Results: Two single guide RNA (sgRNA) candidates were designed, for promoting Cas9 cleavage in the s100A gene cluster in CHO-K1 cells. The cleavage efficiency of both sgRNA were analyzed and sgRNA2 was selected for Cris-PITCh mediated integration of the transgene cassette into the s100a gene cluster in the CHO-K1 cell genome. Nocodazole chemically interferes with the organization of microtubules and causes cell arrest in G2/M phase. In this study 31 hours after the concomitant delivery of Cas9/sgRNA and donor plasmid. Cells were treated with nocodazole for 15 hrs to accumulate cells in the G2/M phase and avoid the following replication cycle. We applied flow cytometry to analyses distinct populations of cells in the different cell cycle and indicated that nocodazole treatment, arrested about 85% of cells in G2/M phase. When nocodazole was removed, a stable cell pool was generated by puromycin selection of transfected cells. limited dilution was done and we obtained 72 recovered single clones in Nocodazole-treated group and 33 recovered single clones in control group (without nocodazole treatment), among which 72 clones of nocodazole group, 51 were 5’/3’ junction positive and among 33 clones in control group, 9 were 5’/3’ junction positive. These results show that targeted transgene knock-in was successfully achieved. Knock-in efficiency for nocodazole group was 64% and for control group was 27%.
  • Conclusion: In this study we have successfully accompanied Crispr/cas9-based MMEJ with a (nocodazole) small molecule for improvement of Cris-PITCh knock-in efficiency. Here, we observed a marked increase (about 2.37 fold) in MMEJ activity. According to the previous studies HR-mediated repair pathway was gradually shut down when cells exited S phase. This finding suggests that these cells don’t use HR pathway, but the MMEJ pathway remains available to fix DSB in this cells.
  • Keywords: Cris-PITCh; small molecule; CRISPR/Cas9 system; MMEJ repair pathway