Barcoding Cells Using Cell-Surface Programmable

We develop here a novel approach to barcode large numbers of cells through cell-surface expression of programmable zinc-finger DNA-binding domains (sZFs). We show sZFs enable double-stranded DNA to sequence-specifically label living cells, and also develop a sequential tagging approach to in situ image >3 cell types using just 3 fluorophores. Finally we demonstrate their broad versatility through ability to serve as surrogate reporters and facilitate selective cell capture and targeting. The ability to construct and interrogate complex tissues and cellular libraries at single cell resolution requires methods that enable highly multiplexed in situ probing of living cells. Here while the use of fluorescent proteins has revolutionized probing of biological phenomena, their multiplexed use is limited to combinations that can be spectrally resolved. To expand the repertoire of probing tools, we explored the possibility of using DNA binding domains such as zinc finger proteins (ZFs) and transcription activator-like effectors (TALEs). Our motivation stemmed from the observation that as a receptor-ligand pair the ZF-DNA or TALE-DNA interaction is very unique in that both the receptor (ZF or TALE protein) and the ligand (DNA) are highly programmable, and hence the space of engineerable orthogonal interactions is huge. Consequently they can be leveraged for engineering macromolecular interactions beyond genome targeting1–4. Specifically, here we exploit the programmability of this interaction to devise a scheme to barcode and image large numbers of cell types by anchoring zinc finger proteins to the outside of the cell membrane and thus making them accessible to DNA based probes provided in the extracellular medium. To express zinc-finger DNA binding domains on the cell surface, we fused at their Nterminus an Ig κ-chain leader sequence and at the C-terminus a platelet derived growth factor (PDGF) transmembrane domain (refer Methods)5. To test the ability of surface zinc finger (sZF) expressing cells to bind DNA we exposed them to fluorophore tagged DNA molecules. sZF expressing cells strongly bound the DNA while control cells exhibited very low binding signals, implying functional zinc-finger proteins were successfully expressed on 4Correspondence should be addressed to gchurch@genetics.med.harvard.edu. 3These authors contributed equally to this work. Author Contributions PM and GMC conceived the study and designed the experiments. PM performed experiments. JL, DL and LN developed reagents. JA developed the image analysis suite and performed associated analyses. PM, JA and GMC wrote the manuscript with support from all authors. NIH Public Access Author Manuscript Nat Methods. Author manuscript; available in PMC 2013 November 01. Published in final edited form as: Nat Methods. 2013 May ; 10(5): 403–406. doi:10.1038/nmeth.2407. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript the cell surface (Fig. 1a). Two aspects of this sZF-DNA interaction were of note: First, sZFs were observed to bind to both single6 and double stranded DNA molecules (Supplementary Fig. 1a), however the former interaction was abrogated in the presence of competitor dsDNA (here Salmon Sperm DNA). Second, sZFs also non-specifically bound to dsDNA, but again in the presence of competitor dsDNA binding to only their cognate target dsDNA was retained (Supplementary Fig. 1b). Similar results were obtained using FACS based assays too (Supplementary Fig. 2). Thus in the presence of competitor dsDNA, sZF expressing cells specifically bind their target dsDNA probe and hence each zinc-finger protein uniquely barcodes the cell type expressing them (Fig. 1b). A total of 16 zinc finger proteins7 were tested using this approach (protein sequences and target dsDNA sequences are provided in Supplementary Table 1). Several aspects of sZFdsDNA interactions emerged from this analysis. First, different sZFs have different binding affinities for their target dsDNA (Fig. 1c). Specifically, while some bound, as assayed by both fluorescence intensity and duration of binding, their targets strongly (ZFs 1, 3, 8, 12, 13, 15, 16), some were moderately strong binders (ZFs 2, 4, 5, 6, 7, 10, 14), while others were only weak binders (ZFs 9, 11). Next we evaluated the sZF cross reactivity profile for these 16 ZFs (Fig. 1d). We found that while most zinc fingers bound their target dsDNA specifically, some showed a significant degree of cross-reactivity (ZFs 1, 8, 13). The strong ZF binders were particularly susceptible to this phenomenon8. Interestingly, almost all the zinc fingers were observed to bind the ZF16 target dsDNA, likely in part to the high poly-G rich content of this sequence. Based on the above ZFs 2, 3, 4, 5, 6, 7, 10, 12, 14, 15 were found to be orthogonal to each other and were moderate to strong binders and thus good candidates for barcoding cells. If sZFs are to serve as efficacious barcodes compatible with analysis of structured tissues, they must enable differential labeling of cells in complex mixtures that is detectable in microscopic images. To investigate this we designed experiments to image and analyze mixtures of sZF expressing cell populations. Specifically, cells expressing either sZF1, sZF2, sZF3 or sZF4 were mixed in pairs (sZF1+sZF2; and sZF3+sZF4) or in a pool of three (sZF1+sZF2+sZF3), and were probed using appropriate combinations of fluorophore labeled target dsDNA molecules. We then developed a suite of MatLab GUI applications to analyze the resulting images and compute quantitative measures of the specificity of binding of sZFs to their corresponding oligos at both the whole cell and single pixel level (processing flow for images is depicted in Supplementary Fig. 3). Qualitative inspection and quantitative analysis of the images confirms that the sZF-dsDNA interactions are sequence specific (Figs. 2a, 2b, & 2c, Supplementary Table 2, and Supplementary Figs. 3–9). Exploring additional ZFs, or extending this approach to TALEs9, 10 will further expand and refine the list of orthogonal interaction pairs that can be exploited for cellular barcoding. Regardless, one is still limited by the small number of spectrally distinct fluorophores available for simultaneous cell imaging. To address this problem we next devised a sequential live-cell hybridization and imaging approach (suitable for adherent cells). It uses a modified two-part DNA probe that presents a double-stranded portion that binds the sZF and a single-stranded portion containing barcode sequences that can be read-out by serial hybridizations (approach in Fig. 2d): this approach is fast and does not use enzymes or chemical reactions and is thus compatible with use on live cells. Extending this scheme to n steps enables barcoding of 3n cell types using just 3 fluorophores. A basic demonstration of the scheme in a simplex setting is provided in Fig. 2e where sZF expressing cells are sequentially probed each sZF identity here is encoded by two colors, for instance sZF2 by green in step 1 and red in step 2, sZF3 by red in step 1 and blue in step 2 and similarly for sZFs 6, 12, 14 and 15 (Fig. 2f). We were also able to mix up to six individually labeled cells and identify their barcode in situ using two hybridization cycles (Figs. 2g, 2h). In these Mali et al. Page 2 Nat Methods. Author manuscript; available in PMC 2013 November 01. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript experiments, the zinc finger-binding probes were also re-supplied for each round of sequencing by hybridization. This re-probing compensated for the loss of fluorescence signal due to the dissociation of dsDNA probes from the sZFs in the interval between imaging steps, and also aided in active displacement of the existing probes, thus mitigating effects of any incomplete quenching in the previous step (refer also Supplementary Figs. 10, 11 respectively for the dissociation kinetics of dsDNA probes and confirmation of the genotype-to-labeling association in these experiments). Use of toe-hold mediated strand exchange11 to displace bound DNA probes can be exploited to further refine this technique. Overall, our results in Fig. 2e and Fig. 2h above suggest that such a sequential tagging scheme can successfully identify the various constituent cells in complex mixtures of barcoded cell types using just 3 spectrally distinct fluorophores. Finally we explored the versatility of sZFs through three applications. First we used sZFs as surrogate reporters of endogenous cellular activity. For this, lentiviral vectors with small molecule (tetracycline and cumate) inducible promoters for driving sZF expression were constructed. Stable transductions of 293T and HeLa cells were performed, and upon small molecule induction sZF expression could indeed be readily detected by the ability of the cells to bind dsDNA molecules (Fig. 3a, Supplementary Fig. 12). This temporally inducible expression of barcodes can also be exploited to minimize effects of sZF expression on cell physiology and toxicity (Supplementary Fig. 13). Second, we exploited the fact that sZFs are expressed on cell surfaces where they are physically accessible and can thus provide convenient handles for DNA mediated cell capture. Specifically, sZF expressing cells were successfully enriched from a mixed population of K562 cells by performing a pull-down using either dsDNA probe-conjugated magnetic beads (Fig. 3b), or on dsDNA arrays (Supplementary Fig. 14). Third, we demonstrated sZF mediated selective gene delivery by pseudotyping12 lentiviruses with dsDNA probes (Supplementary Fig. 15). Specifically, these modified lentiviruses successfully delivered genes to sZF barcoded cells, but in the absence of the DNA pseudotyping, lentiviral delivery efficiency was significantly diminished (Fig. 3c). Taken together, these three applications demonstrate that sZFs have uses beyond direct labeling that include state-probing, capt