Tuneable endogenous mammalian target complementation via multiplexed plasmid-based recombineering.

Recent work provided evidence that quantitative rather than qualitative differences in the proteomic inventory between different cell types and tissues are the cause of functional differences1,2,3,4,5,6,7. Thus, to investigate cell type-specific quantitative consequences of disease mutations or alternative spliced variants, it is of crucial importance to combine knockdown of the endogenous protein(s) with the precise regulated expression of the mutant protein(s) to the desired physiological level. Gene silencing based on short interfering RNAs (siRNA) has been demonstrated in several organisms, including in cultured mammalian cells8, and works by targeting complementary messenger RNA (mRNA) thereby activating the RISC complex causing sequence-specific mRNA degradation. Short hairpin RNAs (shRNAs) can also be stably expressed downstream of polymerase III (Pol III) promoters9 or bidirectionally co-expressed10. Advanced genome editing technologies such as meganucleases, zinc finger nucleases, TALENS (protein-DNA guided), and the RNA-guided CRISPR-Cas9 and CRISPRi-dCas9 methods complement the available tools for gene silencing, each with their own merits11,12. However, despite these being powerful tools, they could modify the genome irreversibly and have other drawbacks. These may include off-target mutagenesis, although in some cases they have been significantly diminished13, and often cumbersome and time-consuming selection/screening to identify the appropriate clone, which is refractory to high-throughput14. Plasmid-based gene delivery systems have become essential for molecular and cell biology. However, the number of available selection markers and the physical space that needs to be available to allow entry of multiple plasmids into cells are technically limiting. These problems are exacerbated by imbalanced delivery if multiple plasmids are used, resulting in heterogeneous cell populations which interferes with read-out. These impediments can be circumvented by combining independent modules containing genes of interest, regulatory elements and other desired functionalities into a single multifunctional multigene delivery plasmid. This is a viable option if the combination of the modules is sufficiently straight-forward. Addressing this challenge, we previously developed the ‘ACEMBL’ system to enable fast and flexible generation of multigene delivery constructs by an automatable technique called tandem recombineering (TR) to combine ‘Donor’ and ‘Acceptor’ plasmid modules via reversible Cre-loxP fusion in vitro, optionally in high-throughput by robotics15,16. We implemented ACEMBL successfully for complex expressions in prokaryotic and eukaryotic cells. However, a useful tool that would afford the means to combine efficient DNA delivery with regulated heterologous expression and efficient silencing in cell-based complementation assays remained elusive to date. Here, we introduce TEMTAC, a novel system for Tunable Endogenous Mammalian TArget Complementation by multiplexed plasmid-based recombineering. Retaining the original ACEMBL concept, we developed this novel system which affords the means to simultaneously knockdown endogenous targets via RNA interference8,9,10 and express mutated versions utilizing regulated tetracycline (TET) controlled transcriptional activation17. Three modules are combined, one each for (i) shRNA-mediated silencing, (ii) TET-regulated expression of the mutant gene of interest (GOI) that contains silent mutations to forestall its own degradation, and (iii) integration of the complete composite plasmid, which expresses an additional YFP marker gene for identifying transfected cells by fluorescence (Fig. 1a). The three modules can be easily assembled and disassembled in vitro exploiting the Cre-Lox recombination reaction, which can be carried out optionally in high-throughput by robotics applying automated routines we have implemented16. The separate modules also work autonomously, for example to test proper functioning of each individual element prior to multigene recombineering. A homing endonuclease (HE)/BstXI based multiplication module15 is provided to enable engineering within each Donor or Acceptor, if several proteins, for example entire regulatory cascades, are to be complemented concomitantly. As a further option, TEMTAC also affords stable integration into cellular genomes, by providing on the Acceptor module eukaryotic resistance markers and the DNA elements required. We demonstrate the utility of TEMTAC utilizing proteins of the ErbB signalling network, HRAS, BRAF, and SHP2. By using TEMTAC, we silence the proteins of interest, express corresponding mutants, and test these for signalling phenotypes, compellingly validating our approach. Figure 1 TEMTAC system components.