• Increasing CRIS-PITCh Method of Gene Editing Efficiency by Using RNP System Delivery
  • Setare Adibzadeh,1 Shahin Amiri,2 Faezeh Feghhi,3 Farzaneh Barkhordari,4 Fatemeh Davami,5,*
    1. Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
    2. Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
    3. Cellular and Molecular Research Center, Iran University of Medical Sciences
    4. Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
    5. Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.


  • Introduction: The main workhorses for producing recombinant therapeutic proteins with complicated glycoforms are Chinese hamster ovary (CHO) cells. Recombinant CHO (rCHO) cell lines are typically created by randomly integrating a gene of interest (GOI) into the genome, then selecting cells that carry the transgene. Lack of control over gene insertion, on the other hand, might result in undesirable phenotypic variability because of the variable accessibility of integration sites for gene expression, also known as position effect variation. These cell lines are hence frequently unstable and gradually exhibit reduced production. To choose the right clones suitable for high and steady expression of recombinant proteins, further screening of several clones is required because of this heterogeneity in expression and genomic composition. Recent releases of the draft genomes of multiple CHO cell lines have made it possible to effectively modify the genomic sequence of CHO cells using engineered nucleases. The CRISPR/Cas9 platform's foundation is a simple base-pairing interaction between an engineered RNA and the targeted genomic site, allowing for quick design, simple use, and low costs. The target locus will normally be repaired by one of the two main DNA damage repair pathways following site-specific DNA double-strand breaks (DSBs) carried on by designed nucleases: the error-prone nonhomologous end-joining (NHEJ) or non-efficient homology-directed repair (HDR) which use long homology arms. An alternative end-joining path called microhomology-mediated end-joining (MMEJ) uses microhomology arms (20–40 bp), which are active during most phases of the cell cycle. Short arms make the donor design simple and economical. In order to use the MMEJ mechanism in CRISPR-mediated knock in, the CRIS-PITCh (Precise Integration into Target Chromosome) technology has recently been created. The short homology arms of this system's transgene are flanked by sgRNA target sequences, which are used to linearize the donor in vivo and release the transgene. However, the knock-in efficiency of CRIS-PITCH is low. In recent years, attempts have been undertaken to improve CRIS-PITCH's efficiency inside the cell. Plasmid DNA (pDNA), messenger RNA (mRNA), or ribonucleoprotein (RNP, Cas9 protein complexed with sgRNA) are all possible means of delivery for the CRISPR/Cas9 complex. The fastest genome editing is possible thanks to RNP delivery because it does not require intracellular transcription and translation. Transient genome editing, on the other hand, not only enables excellent editing efficiency but also minimizes immunological reactions, insertional mutagenesis, and off-target consequences. The PITCH-CRISPR system in the high-producing cell line CHO-K1 has not been made more effective using this technique. Using this study, we knock-in a landing Pad (LP) with RNP method, the problem of the low efficiency of the PITCH-CRISPR system will be solved to some extent.
  • Methods: Donor plasmid for the LP construct contained HSV-TK, T2A, and puroR expression units flanked by 30 bp microhomology arms and PITCh gRNA cut sites. For plasmid based transfection an all-in-one plasmid containing tandem U6-PITCh gRNA, U6-genome targeting gRNA, and Cas9 nuclease was used. the single-stranded oligos for PITCh gRNA and Genome-targeting gRNAs synthesized according to Gene Art Precision gRNA Synthesis Kit. Before RNP complex transfection the cells were transfected with the LP donor vector using Lipofectamine 3000 reagent according to the manufacturer’s protocols. Two RNP complexes were formed, one containing PITCH gRNA and the other containing genomic target. Transfection with Lipofectamine CRISPRMAX was performed according to manufacturer’s protocols. In order to plasmid based assay the cells were cotransfected with the Cas9-sgRNAs vector and LP donor vector using Lipofectamine 3000 reagent. Following puromycin selection, the cell pools were harvested, and genomic DNA was extracted, 5’/3’ junction PCR analysis was performed. The intact integration of a transgene was also confirmed with the out-out PCR. The clonal selection was performed using the limiting dilution method. then, PCR analysis was performed on the genomic DNA of each single-cell clone. The relative copy number of integrated thymidine kinase (TK) in single-cell clones was analyzed by quantitative real-time PCR
  • Results: In this study, by using RNP, we were able to increase the efficiency of the PITCH-CRISPR system in the s100 locus of the CHO-k1 genome by 43% comparing plasmid based CRISPR strategy (22%).
  • Conclusion: Using this study, we knock-in a landing Pad (LP) with RNP method, so the problem of the low efficiency of the PITCH-CRISPR system will be solved to some extent.
  • Keywords: CRISPR/Cas9, MMEJ , CRIS-PITCh, sgRNA, CHO cells