SABER Enables Highly Multiplexed and Amplified Detection of DNA And

bioRxiv preprint doi: https://doi.org/10.1101/401810; this version posted August 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues Jocelyn Y. Kishi1;2;† Brian J. Beliveau1;2;† Sylvain W. Lapan3;† Emma R. West3 Allen Zhu1;2 Hiroshi M. Sasaki1;2 Sinem K. Saka1;2 Yu Wang1;2 Constance L. Cepko3;∗ Peng Yin1;2;∗ Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 021151 Department of Systems Biology, Harvard Medical School, Boston, MA 021152, Department of Genetics, Harvard Medical School, Boston, MA 021153, These authors contributed equally†, Correspondence∗: [email protected] (P.Y.), [email protected] (C.L.C.) SUMMARY of quantitative FISH signals. These strategies include the targeted deposition of detectable reactive molecules around the site Fluorescent in situ hybridization (FISH) reveals the abun- of probe hybridization,11–14 the targeted assembly of ‘branched’ dance and positioning of nucleic acid sequences in fixed sam- structures composed of DNA15–17 or locked nucleic acid (LNA) ples and can be combined with cell segmentation to produce molecules,18 the programmed in situ growth of detectable con- a powerful single cell gene expression assay. However, it re- catemers by enzymatic rolling circle amplification (RCA)19,20 mains difficult to label more than a few targets and to visu- or hybridization chain reaction (HCR),21–25 and the assembly of alize nucleic acids in environments such as thick tissue sam- topologically catenated DNA structures using serial rounds of ples using conventional FISH technologies. Recently, meth- chemical ligation.26 ods have been developed for multiplexed amplification of Using conventional FISH approaches, it is also difficult to FISH signals, yet it remains challenging to achieve high lev- examine more than a few targets in the same sample due to the els of simultaneous multiplexing combined with high sam- relatively small number of spectrally distinct imaging channels pling efficiency and simple workflows. Here, we introduce available on fluorescent microscopes. In order to overcome this signal amplification by exchange reaction (SABER), which limitation, methods have been introduced that rely on creating endows oligo-based FISH probes with long, single-stranded additional ‘colors’ by labeling the same target with multiple DNA concatemers that serve as targets for sensitive fluores- fluorophores in defined combinations27,28 and ratios,29 allow- cent detection. We establish that SABER effectively ampli- ing the unambiguous detection of all 24 human chromosomes fies the signal of probes targeting nucleic acids in fixed cells on metaphase spreads27–29 and in interphase nuclei7 and >30 and tissues, can be deployed against at least 17 targets si- mRNA targets in budding yeast.30 Additional approaches have multaneously, and detects mRNAs with high efficiency. As been introduced that utilize serial rounds of imaging, label re- a demonstration of the utility of SABER in assays involv- moval, and re-labeling of distinct targets, enabling researchers ing genetic manipulations, we apply multiplexed FISH of to image potentially unlimited numbers of targets. These strate- reporters and cell type markers to the identification of en- gies have been used to visualize up to 40 distinct chromosomal hancers with cell type-specific activity in the mouse retina. regions in tissue culture cells by imaging each target region just SABER represents a simple and versatile molecular toolkit once with a single fluorophore.31 Multi-round combinatorial la- to allow rapid and cost effective multiplexed imaging. beling strategies, which allow an exponential number of unique, non-overlapping targets to be visualized in a linear number of INTRODUCTION rounds, have been used to image 12 mRNA targets in budding yeast,32 >1000 mRNA targets in tissue culture cells,33 and in- Fluorescent in situ hybridization (FISH) allows researchers tronic sequences34 of >10,000 nascent RNAs in tissue culture to interrogate the subcellular distribution of RNA and DNA cells.35 However, because these strategies require spatially sep- molecules in fixed cells and tissues. First introduced in the late arated targets, only much more modest multiplexing levels have 1960s, in situ hybridization methods1 and their fluorescent vari- been demonstrated with arbitrarily dense targets.36 In principle, ants2–5 utilize detectable nucleic acid probes that can be pro- all of these multiplexing strategies could benefit from signal am- grammed to hybridize to complementary cellular targets. FISH plification, which could reduce requirements on expensive mi- assays are integral to many fields and are used for diverse appli- croscopy setups, shorten imaging times, and potentially reduce cations such as the diagnostic detection of chromosomal abnor- cost by lowering the number of probes required. This ampli- malities,6 the interrogation of three-dimensional genome orga- fication would be particularly relevant in the context of thick nization,7,8 and the quantitative analysis of gene expression.9,10 tissues, which can suffer from high levels of autofluorescence, Despite their widespread use, conventional FISH approaches light scattering, and optical aberration that can make signal de- are still limited by technical considerations. For instance, it tection challenging. can be challenging to visualize hybridization events in com- Excitingly, recent studies have begun to integrate serially plicated environments such as tissue samples due to inefficien- multiplexed imaging with signal amplification to detect targets cies in probe penetration and light collection. Accordingly, sev- in tissue samples. One approach, seqFISH,32 combined HCR eral approaches have been developed to amplify the intensity with a combinatorial multi-round labeling strategy to visualize bioRxiv preprint doi: https://doi.org/10.1101/401810; this version posted August 27, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. up to 249 mRNA targets in 15µm mouse brain tissue sections.37 DNA and RNA FISH probes are first chemically synthesized However, design of orthogonal HCR concatemers is difficult with primer sequences on their 3’ ends and then concatemer- due to the challenge of designing multiple non-interfering pro- ized using PER in vitro. Extended probe sequences are then grammed reaction pathways to operate in situ, and only 5 or- hybridized in situ and act as scaffolds to which multiple fluores- thogonal HCR concatemers have been demonstrated. As a recent strands can bind. We establish that SABER provides a fast, sult, only 5 barcodes could be deployed simultaneously in each simple, and inexpensive platform to amplify the signal of both sequential round, with each round consisting of concatemer di- RNA and DNA FISH probes in fixed cells and tissues. We exper- gestion (4 hours), probe hybridization (overnight), amplification imentally demonstrate that in different scenarios, SABER can (~1 hour), and imaging.37 Another approach, STARmap, used a amplify a signal up to 450 fold, can be deployed against up to 17 novel form of RCA coupled with in situ RNA sequencing38 to different targets simultaneously, and can provide high sampling detect up to 28 mRNA targets simultaneously in cleared 150µm efficiency of target transcripts for puncta detection and cell type mouse tissue sections and up to 1,020 spatially separated tar- identification. We further highlight an application of SABER in gets in thin (single cell layer) sections.39 While RCA-based tissues by applying a novel 10-plex FISH assay for interrogat- methods in principle overcome the limitation of HCR by en- ing the activity and specificity of candidate enhancer elements abling highly multiplexed simultaneous amplification, detection in mouse retinal tissue via the detection of reporter RNAs, fol- efficiencies for RNA transcripts remain comparable to single- lowed by co-detection of reporters and plasmids through a com- cell RNA sequencing (6-40%) in the best case (STARmap) and bined RNA/DNA FISH experiment. are even lower for other RCA-based methods such as FISSEQ (0.01-0.2%) and padlock probe-based designs (5-30%),39 per- RESULTS haps due to the complexity of controlling parallel enzymatic re- actions in situ. Furthermore, strategies combining signal ampli- Design of orthogonally detectable sequences for use in build- fication and high levels of multiplexing remain challenging for ing probe-linked concatemer structures many labs to implement, particularly in preserved tissue, in part We recently developed the primer exchange reaction (PER) due to the complexity and cost of existing technologies at the method for autonomously synthesizing arbitrary single-stranded level of probe design, tissue pre-treatment, method workflows, DNA sequences from short DNA primers.40 The method can limitation to spatially separated targets, image registration, and be used to programmably append specific DNA sequence do- computational alignment. Accordingly, we set out to create a mains onto growing single-stranded concatemer sequences. One quantitative FISH method

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