Design and construction of a universal payload vector for bxb1-based recombinase-mediated cassette exchange for targeting a transgene into a single locus in mammalian cell

Design and construction of a universal payload vector for bxb1-based recombinase-mediated cassette exchange for targeting a transgene into a single locus in mammalian cell
Samaneh Ghanbari,¹ Fatemeh Davami,^2,*
1. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
2. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
Introduction: Chinese hamster ovary (CHO) cells are the most commonly used host for biotherapeutic production. The traditional method for stable cell line development is based on random integration of a gene of interest (GOI) which needs screening of several hundred clones to obtain high producer ones. With the advent of new genome-editing systems such as the CRISPR/Cas9 system, a new approach is toward site-specific integration which reduces clonal variation and accelerates the cell line development process. Recombinase mediated cassette exchange (RMCE) utilizes a site-specific recombinase/integrase which mediates recombination between its pre-integrated recognition site into a genomic site and its cognate attachment site on a transgene harboring vector (payload vector). It takes up to 12 months from transfection to lead clone cell banking in traditional cell line development. By using the CRISPR/Cas9 system in conjunction with RMCE technology (hybrid system), cell line development timelines can be significantly reduced to 4 weeks. In the hybrid system, CRISPR/Cas9 can be used to generate RMCE founder clone, a clone which contains a recombinase recognition site (attP site for bxb1 recombinase) in a specific locus. After characterization of the founder clone, it can be used repeatedly. In the presence of a recombinase vector, the landing pad of the founder clone is swapped for a therapeutic transgene using RMCE. To shorten the time frame even more, we have designed and constructed a universal payload vector with a multiple cloning site flanked by bxb1 recombinase recognition site (attB site). We also incorporated the Herpes Simplex Virus-1 thymidine kinase (HSV-TK) cDNA in the donor vector backbone to eliminate any possible random integration event following ganciclovir selection.
Methods: Construct design: Bx1 attB site sequence was obtained from literature review, to prevent unintended integration of plasmid backbone instead of donor cassette the multiple cloning site (MCS) flanked by inversely oriented attB sites (attB and attBi). Since orthogonal recombinase target site is needed in RMCE system, the cassette consist of MCS flanked in one side by bxb1 attB-GA mutant, and in the other side by the wild type one. To be able to clone HSV-TK cDNA, a cloning site was considered outside the attB sites. The designed construct was synthesized by Bio Basic Inc. (Markham, Ontario, Canada). HSV-TK cloning and transformation HSV-TK cassette together with its promoter PGK was PCR amplified from an in-house plasmid using forward and reverse primers containing XbaI and KpnI site, respectively. XbaI/ KpnI digested PCR product was ligated into the XbaI/KpnI linearized payload vector. The ligation product was transformed into chemically competented E. Coli TOP10F´strain. Cloning confirmation Colony PCR was performed to screen colonies for the desired insert. The plasmid was extracted from a positive clone, digested with XbaI and KpnI, and analyzed by gel electrophoresis. Finally, the plasmid DNA was sent for sequencing (Macrogen, Korea).
Results: Schematic map of the designed plasmid containing attB-MCS-mutattBi and HSV-TK cassette is shown in Fig. 1. To verify cloning of PGK-TK into XbaI/KpnI site, resulting colonies were screened by colony PCR using forward primer on plasmid backbone and reverse primer on PGK construct leading to a 655 bp amplicon. Gel electrophoresis of PCR products shows 5 out of 5 clones were positive for cloning (Fig.2). An extracted plasmid from a positive clone was then digested with XbaI and KpnI. Fragments were verified by gel electrophoresis (Fig. 3).
Conclusion: In summary, we designed and constructed a universal payload vector to be used in CRISPR/cas9-RMCE hybrid system for targeted integration of a transgene into a defined locus in mammalian cells. By implementation of inversely oriented bxb1 attB sites, the possible integration of the vector backbone instead of the gene of interest has been eliminated. HSV-TK cassette on the vector backbone allows us to select transfected cell pool with ganciclovir resulting in a homogenous cell pool without random integrants. This ready-to-use payload vector only requires GOI cloning so reduces the time, burden and cost of multiple fragments cloning or synthesis.
Keywords: CRISPR/Cas9, recombinase-mediated cassette exchange, targeted integration, cell line development