Split Selectable Markers

Selectable markers, such as antibiotic resistance or fluorescent protein genes, are widely adopted in transgenesis and genome editing for selecting engineered cells with desired genotype but are limited in choices. We demonstrate here the concept of split selectable markers each allowing for selection of multiple “unlinked” transgenes with one selection scheme. Future development of split selectable markers may support enrichment or selection of “hyper-engineered” cells containing tens of transgenes or genetic modifications.

2 ("markertrons") that can be rejoined by protein trans-splicing 3 (Fig 1a). Each markertron is inserted onto a transgenic vector carrying a specific transgene. Delivery of transgenic vectors containing a set of markertrons yield cells harboring a subset or a complete set of the marketrons. Only cells containing a complete set of markertrons produce a fully reconstituted marker protein via protein splicing and thus passes through selection while cells with partial sets of markertrons are eliminated, achieving co-selection of cells containing all intended transgenes.
We started out with engineering 2-markertron intein-split resistance (Intres) genes for double transgenesis. Since flanking residues and local protein folding can affect efficiency of intein-mediated trans-splicing, we set out to identify split points in each of the four commonly used antibiotic resistance genes compatible with two well-characterized split inteins derived from NpuDnaE 4,5 and SspDnaB 6 . To facilitate assessment of the effectiveness of double transgenic selection, we cloned markertrons onto lentiviral vectors expressing TagBFP2 or mCherry fluorescent proteins as test transgenes (Fig 1b). Viral preparations were transduced into U2OS cells which were then split into replicate plates with nonselective or selective media. Following appropriate passages for antibiotics selection, the two cell cultures were analyzed by flow cytometry. For hygromycin (Hygro) resistance gene, one "native" SspDnaB split point (G200:S201) with flanking residues "GS" and one "native" NpuDnaE split point (Y89:C90) with "YC" residues were tested. Both enabled successful selection when both N-and C-markertrons were transduced yielding >99% BFP+ mCherry+ double transgenic cells in selected cultures compared to <10% double-positive cells in non-selected culture (Fig 1c; Plasmid pairs 3,4 and 5,6). Cells transduced with either of the two markertrons did not survive hygromycin selection. In contrast, double transgenesis with conventional full-length non-split hygromycin vectors only allow for ~20% enrichment of BFP+ mCherry+ cells (Plasmid pairs 97,98). We screened three additional potential split points (52S:53C),(240A:241C), and (292R:293C) for NpuDnaE with the obligatory cysteine residue on the Cextein junction and a residue on the N-extein junction that supported substantial trans-splicing activities in a previous report 7 . We also incorporated six additional NpuDnaE split points by inserting an "artificial" cysteine on the C-extein junction to support splicing at ectopic sites yielding additional split points. In total, eight out of eleven split points tested supported hygromycin selection (Fig 1c). Similarly, for puromycin 3 (Puro) (Fig 1d), neomycin (Neo) (Fig 1e) and blasticidin (Blast) (Fig 1f) resistance genes, we identified four, two, and one functional Intres pair(s), respectively. In all of these cases, cells transduced with either markertrons did not survive selection, while cells transduced with both yielded >95% double transgenic cells in selective cultures compared to <50% in non-selective cultures with the exception of Blasticidin(102) Intres, achieving lower but still significant enrichment of 91% double transgenic cells (Fig   1c~f). To facilitate adoption of Intres markers, we created Gateway-compatible lentiviral vectors for convenient restriction-ligation-independent LR clonase recombination of transgenes 8 (Fig 2a). We tested the functionality of these vectors by recombining TagBPF2 and mCherry, respectively to the N-and C-Intres vectors and found robust selection of double transgenic cells (Fig 2b). One potential utility of Intres vectors is to install different fluorescent markers in cells to label different cellular compartments. To explore such utility, we cloned in NLS-GFP and LifeAct-mScarlet 9 , which label nucleus and F-actin, respectively, by Gateway recombination to conventional full length (FL) non-split hygromycin selectable vectors or 2-markertron hygromycin Intres vectors and transduced cells with either sets of plasmids, followed by antibiotic selection (Fig 2c). The sample transduced with non-split selectable plasmids contained both singly and doubly labelled cells, while cell transduced with Intres plasmids were all doubly labelled (Fig 2c). Details of the split points of Intres genes and plasmids are presented in Supplementary   fig 1~4 and Supplementary table 1. To test whether split fluorescent markers can be used for transgene selection, we screened for NpuDnaE split points for mScarlet fluorescent protein (Fig 3a) and identified four split points allowing for >96% enrichment of double transgenic cells and three other split points enabling >60% enrichment of double transgenic cells in mScarlet-gated population, compared to <20% double transgenic cells in non-gated population (Fig 3b).
With the split points identified for 2-markertron Intres genes, we set out to engineer higher degree split markers. We tested combinations of splits points to partition a marker gene into three or more markertrons to allow for co-selection of more than two "unlinked" transgenes with one antibiotics (Fig 4a   and b). To identify pairs of split points that would allow such "Intres chain", we cloned 3-split markertrons 4 into three lentiviral vectors each carrying one of three fluorescent transgenes TagBFP2, EGFP, or mCherry, that will allow us to assess effectiveness of selection by flow cytometry (Fig 4c). Since hygromycin resistance gene is the longest and provides the most split points for testing, we focused on engineering 3-markertron hygromycin Intres. We tested two 3-markertron hygromycin Intres using two intervening NpuDnaE inteins, two using NpuDnaE for the first intein and SspDnaB for the second intein, as well as two using SspDnaB for the first intein and NpuDnaE for the second intein (Fig 4d). Five of these six 3-markertron hygromycin Intres enabled >97% and with the remaining one enabling 80% triple transgenic selection in hygromycin-selected cultures compared to <15% triple transgenic cells in nonselected cultures. Samples with leave-one-out transduction did not yield any viable cells after hygromycin selection while cells transduced with non-split hygromycin vectors yielded only 7% triple transgenic cells after selection. To facilitate the use of 3-markertron Intres, we created Gateway compatible lentiviral vectors with these markers (Fig 5a). Three sets of these vectors were each tested by recombining TagBFP (as transgene 1), EGFP (as transgene 2) and mCherry (as transgene 3) into the N-, M-, and C-Intres Gateway destination vectors and used to transduce U2OS cells, which were then split and cultured in hygromycin selection or non-selective media (Fig 5b). Two weeks after selection, cells were analyzed by flow cytometry. All three sets of 3-markertron hygromycin Intres plasmids support triple transgenic cell selection of >99% compared to <25% in the non-selected cultures (Fig 5c). We further tested the feasibility of 4-markertron hygromycin Intres genes ( Supplementary Fig 6a). Here, we used an enhanced variant of NpuDnaE intein known as NpuDnaGEP 10 fused with leucine zipper motifs 11 in combination with the SspDnaB intein. While transduction of all four plasmids containing constituent markertrons produced cells that survived hygromycin selection, leave-one-out transduction did not yield any survival ( Supplementary Fig 6b).
In this study, we have engineered split antibiotic resistance and fluorescent protein genes that can allow selection for two or more "unlinked" transgenes. By inserting unnatural residues at selectable markers, we showed that novel high-efficiency split points can be utilized, expanding the positions available for engineering. We demonstrated that split selectable markers can be incorporated into lentiviral vectors to enable selection of cells with double transgenesis. By combining two or more splits points, we showed that 3-and 4-split markers can be generated to allow higher degree transgenic selection. Future development of even higher-degree split selectable markers may enable "hyper-engineering" of cells containing five or more transgenes.

Acknowledgements
This research was supported in part by an NHGRI grant 1R01HG009900 (to A.W.C). (b) To screen for split points compatible for inteins for an antibiotic resistance gene, we identified potential split points according to the junctional requirement for the type of intein tested, then cloned the corresponding N-terminal and C-terminal fragments to the split intein scaffolds on lentiviral vectors equipped with TagBFP2 or mCherry fluorescent proteins, which serve as our test transgenes to evaluate selection efficiency. These are delivered into cells via lentiviral transduction. The cells were then split into replicate plates, one subjected to antibiotic selection while the other maintained in non-selective media.

Cloning
To generate a test plasmid for each markertron, we first generated a Gateway donor plasmid containing its ORF and then recombine into lentiviral destination vector with TagBFP2  Selectable marker fragments were amplified from plasmids containing these markers. Plasmids created in this study are listed in Supplementary Table 1. Plasmid maps will be available on http://intr.es Plasmid will be deposited to Addgene for distribution.

Virus Production
A viral packaging mix of pLP1, pLP2, and VSV-G were co-transfected with each lentiviral vector into Lenti-X 293T cells (ClonTech), seeded the day before in 6-well plates at a concentration of 1.2x10 6 cells per well, using Lipofectamine 3000. Media was changed 6h after transfection then incubated overnight.
2 28 hour post transfection, the media supernatant containing virus was filtered using 45 µM PES filters then stored at -80C until use.

Transduction
The day prior to transduction, U2OS cells were seeded into 12-well plates at a density of 1.5x10 5

Fluorescence-Activated Cell Sorting
Cells were trypisinized, suspended in media then analyzed on a LSRFortessa X-20 (BD Bioscience) flow cytometer using FACSDiVa software, version 8, on an HP Z230 workstation. Fifty thousand events were collected each run.