A programmable sequence of reporters for lineage analysis

Abstract

We present CLADES (cell lineage access driven by an edition sequence), a technology for cell lineage studies based on CRISPR–Cas9 techniques. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a predetermined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, thereby coupling birth order to reporter expression. This system, which can also be temporally induced by heat shock, enables the temporal resolution of lineage development and can therefore be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, predominantly marking each generation with the corresponding combination of reporters. CLADES therefore offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.

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Fig. 1: A conditional gRNA scaffold that is activated by other gRNAs.
Fig. 2: A coupled cascade of gRNAs and reporters.
Fig. 3: CLADES 1.0: two reporters, three colors.
Fig. 4: CLADES 2.0: combining a five-color cascade with GAL4 induction.
Fig. 5: Temporal induction of CLADES in AL lineages.
Fig. 6: CLADES as an event tracker: cascade progression across fly generations.

Data availability

The raw data generated during the current study are available from the corresponding authors upon reasonable request.

Code availability

The custom algorithm used to analyze the sequencing data is available from GitHub: https://github.com/tzuminlee/CLADES_amplicon-analysis.

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Acknowledgements

We thank all members of T.L.’s Lab for their comments and feedback, especially R. Miyares for critical reading and input on the manuscript. We also thank H. Lacin and E. Martin-Lopez for their input on the manuscript. We thank Q. Ren and the Janelia Fly Core for their excellent technical support. We thank our suppliers Rainbow, Genscript and Benchling for their services. We thank the Drosophila Genomics Resource Center, supported by NIH grant 2P40OD010949, for the S2 cell line. We thank the Benito-Sipos Lab (Universidad Autonoma de Madrid, Spain) for fruitful discussions and for kindly providing the Lbe(k)–GAL4 line. Stocks obtained from the BDSC (NIH P40OD018537) were used in this study. We thank C. Di Pietro and K. Miller for administrative support. This work was supported by the Howard Hughes Medical Institute.

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Authors

Contributions

Conceptualization: J.G.-M. and T.L. Methodology: J.G.-M. and T.L. Investigation: J.G.-M., C.-P.Y., K.-Y.K., I.E.-M. and H.-H.Y. Writing (original draft): J.G.-M. and T.L. Writing (reviewing and editing): J.G.-M., C.-P.Y., I.E.-M., M.K. and T.L. Visualization: J.G.-M. Supervision: M.K., H.-H.Y. and T.L. Project administration: M.L. and T.L. Funding acquisition: M.K. and T.L.

Corresponding authors

Correspondence to Jorge Garcia-Marques or Tzumin Lee.

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Competing interests

J.G.-M. and T.L. have filed a patent application (PCT/US2018/042731) based on this work with the US Patent and Trademark Office. The other authors declare no competing interests.

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Peer review information Nature Neuroscience thanks James Gagnon, Simon Hippenmeyer, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Optimization of a conditional gRNA.

14 conditional gRNA variants were tested for potential leakiness (activity in the absence of the trigger gRNA). We examined the ability of the conditional gRNA to activate, via SSA, a specific conditional reporter11 (gRNA-spacer reporter, green) in the absence of the trigger gRNA. Each variant was tested with both Dpn-Cas9 and Actin-Cas9. Given that the target sequence (embedded in the scaffold) in the variant #5 unexpectedly acted as an active gRNA, we included a second reporter (gRNA-target reporter, red) to analyze this activity. From the variant #8 onwards we avoided this activity by inverting the orientation of the target sequence. Note that variant #6 is the least leaky as it shows little activity with Actin-Cas9 and almost no activity at all with Dpn-Cas9. Green/Red, immunohistochemistry for EGFP/mCherry. Gray, nc82 counterstain. See also Supplementary Text. n=10 brains. Scale bar = 50 μm.

Extended Data Fig. 2 Analysis of the DNA repair outcome by Next-Generation Sequencing.

a, Flies bearing Dpn-Cas9 and the trigger gRNA#1 driven by the U6 promoter were crossed to a fly with the conditional U6-gRN[#1]NA#2. Progeny with the three components were analyzed by PCR-amplifying the region flanking the target site and sequencing this amplicon by Amplicon-targeted Next-generation sequencing. b, Most frequent repair outcomes are shown, including SSA that covers about 50% of the reads. n=3 replicates, 30 fly heads per replicate.

Extended Data Fig. 3 A conditional gRNA works efficiently in a model vertebrate (zebrafish).

a, Tol2-plasmids were injected into 1-cell-stage zebrafish embryos along with mRNA for the Tol2 transposase. Zebrafish larvae were then analyzed for fluorescence around 24h after fertilization. Each plasmid encodes for: i) Cas9, ii) an RFP protein (injection control) and iii) a YF[#2]FP reporter for the gRNA#2 activity11. These three proteins share the same ORF and are under the regulation of the ubiquitous promoter Ubi. Downstream of this cassette we also placed the conditional U6-gRN[#1]NA#2. b, In the absence of the trigger gRNA, few to no cells expressed YFP. n=22 fish, 3 independent experiments. c, d, Adding a U6-gRNA #1 to the plasmid triggered a gRNA cascade, resulting in most RFP+ cells to co-express YFP. n=11 fish, 3 independent experiments. Circled numbers represent the order of events occurring in the cascade. Scale bar = 200 μm. e, Percentage of RFP+ cells that express YFP. Center line within the box shows median. The box shows the quartiles of the dataset while the whiskers extend to show the rest of the distribution (inter-quartile range 1.5x). n=22 (control) and 11(experimental) fish, 3 independent experiments.

Extended Data Fig. 4 Uncoupling between the gRNA cascade and the reporter cascade.

a, Top, diagram demonstrating the predicted correct order for conditional reporter and gRNA activation. Bottom, representative example of the correct order for a cascade of gRNAs controlling the activation of multiple reports in trans. Since gRNA#1 is active from the beginning and gRNA#2 requires activation by gRNA#1, the activation of the RFP reporter should precede the activation of the GFP. N=10 brains. b, Example where the activation of the second reporter occurs before the first reporter. In both cases the example corresponds to the lateral lineage in the AL, although this was also observed in other lineages. Green, red and gray, immunohistochemistry for GFP, RFP and nc82 (counterstaining) respectively. In the schemes, circled numbers represent the order of events occurring in the cascade. Scale bar = 15 μm. n=10 brains.

Extended Data Fig. 5 CLADES optimization.

Description of the main steps in the generation of a functional CLADES construct. Construct #4 is the pre-activated (ON cassette) version of construct #3. Construct #11 is the pre-activated (ON cassette) version of construct #12 with minor differences. Constructs #1-5, #7 and #11-12 were tested as transgenic flies. Constructs #6, 8, 9-10 were tested in S2 cells. Green check marks and red ‘X’s indicate constructs that functioned or did not function as expected respectively. See Supplementary Text and Material and Methods for a detailed description of the optimization process.

Extended Data Fig. 6 CLADES control constructs.

a, CLADES construct. In the initial state, no fluorescence is observed as translation stops at the ON cassette. b, Pre-activated version of CLADES. In this case, the ON cassette sequence is the same as the expected SSA repair outcome. Red fluorescence is ubiquitous since translation progresses to the end of the reporter. c, Pre-activated+pre-inactivated version of CLADES. Both the sequence for the ON and OFF cassettes is the same as the expected SSA repair outcome. No fluorescence is observed as the translation stops at the OFF cassette. d, Triggering CLADES 1.0 requires both the trigger gRNA#1 and Cas9 for YPF and RFP fluorescence. e, Only a matching gRNA can trigger each of the CLADES 1.0 reporters. Relevant controls were shown, according to the order in the cascade. Flies bearing Dpn-Cas9 and a U6-gRNA (#1 or #2) were crossed to a fly with CLADES 1.0 (d) or only one of the two CLADES 1.0 constructs (e). Green, red and gray, immunohistochemistry for GFP (YFP), RFP and nc82 respectively. n=12 brain in A-C, 24 brains in D and 11 brains for each case in E. Scale bar = 50 μm.

Extended Data Fig. 7 CLADES 1.0 progresses over larval development.

a, Progression of the CLADES 1.0 cascade over the course of larval development, as triggered by the ubiquitous U6-gRNA#1 trigger. Green, red and gray, immunohistochemistry for YFP, RFP and Dpn respectively. n=10 brains. Scale bars = 50 micrometers. b, Percentage of neuroblasts (n=10 brains, 30 neuroblasts each condition) expressing the different reporter combinations. c, Normalization of the data shown in (b) to the maximum percentage for each combination of reporters. Error bars and areas around the line plot represent a 95% confidence interval. n=10 brains, 30 neuroblasts each condition.

Extended Data Fig. 8 CLADES 2.0 reflects the birth-order of the embryonic lineage 5-6 in the VNC.

a, Schematics representing the organization of the lineage 5-6 in the VNC, labeled with CLADES 2.0 and the lbe(k)-GAL4 driver. The position of the neuroblast near the ventral surface generates a gradient with early-born neurons (green) pushed away towards dorsal positions, late-born neurons (red) located near the ventral surface and mid-born neurons (yellow) occupying intermediate positions. b, Representative example of a lineage 5-6 clone labeled with CLADES 2.0+lbe(k)-GAL4. Maximum intensity projection of multiple confocal planes. Green, red immunohistochemistry for V5 (YFP) and RFP respectively. Scale bar = 5 μm. n=9 clones c, Average depth from the ventral surface for neurons expressing each combination of reporters, both for clones with green, yellow and red or only green and yellow neurons. n=126 neurons, 9 clones in total.

Extended Data Fig. 9 Reconstruction of the lateral and dorsal antennal lobe lineages.

a, b, Raw data acquired in the GH146-GAL4/Dpn-Cas9 experiment, for the lateral (a) and dorsal (b) AL lineage. Each row represents a different clone, with each color showing the corresponding reporter combination. Black squares represent unidentifiable glomeruli. c, d, Top, probability of each neuronal type of undergoing at least one transition in the cascade, for the lateral (c) and dorsal (d) AL lineages. Bottom, number of steps in the cascade undergone by different neuronal types. Points with the same color denote the same value. Horizontal lines represent the average number of steps for each neuronal type. e, f, Real and predicted birth-order, based on the average number of steps for each neuronal type in the lateral (e) and dorsal f, AL lineage. Squares denote neuronal types whose birth-order is correct (green) or incorrect (red) compared to the ground truth. n= 58 (lateral) and 157 (dorsal) clones.

Extended Data Fig. 10 Heat-shock induction of CLADES.

a, Schematic representation illustrating the design of a heat-shock promoter with minimal leaky expression. Top) under no-induction, a standard heat-shock promoter exhibits basal transcription. This activity is sufficient for the translation of enough Cas9 protein to induce the progression of the CLADES cascade in neuroblasts. Bottom) adding a ribozyme in the 5’UTR results in the uncapping of the mRNA, reducing the leaky expression of Cas9 and therefore the number of neuroblasts that aberrantly progresses in the cascade. b, Representative example of larval brains labeled with CLADES 2.0+Dpn-GAL4, under no heat-shock conditions. On the top, Cas9 is expressed driven by a regular heat-shock promoter (HSP-Cas9). On the bottom, Cas9 is expressed from the new design with minimal leaky expression (HSP-Rbz-Cas9). n=25 (HSP-Cas9) and 37 (HSP-Rbz-Cas9) larval brains c, Number of yellow or red neuroblasts per brain under no-induction conditions, both for the HSP-Cas9 and HSP-Rbz-Cas9 designs. Horizontal lines represent the corrected average of neuroblasts per brain, with yellow neuroblasts counting as 1 and red neuroblasts as 2. n=25 (HSP-Cas9) and 37 (HSP-Rbz-Cas9) larval brains. d, Cartoon illustrating the induction experiment for the Hsp-Rbz-Cas9 design. After a single heat-shock, neuroblasts progress a single step in the CLADES cascade without the ribozyme compromising the inducibility of Cas9. e, Representative examples of larval brains labeled with CLADES 2.0+Dpn-Gal4 after a single heat-shock at various stages or a double heat-shock at 8-12h AEL+48-60h ALH. n= 48 (8-12h AEL), 30 (36-48h ALH), 25 (48-60h ALH) and 24 (double) brains. f, Number of yellow or red neuroblasts per brain under the different heat-shock conditions. Horizontal lines also represent the corrected average as shown above. g, Raw data acquired in the GH146-GAL4/heat-shock experiment. Rows represent clonal events, with each color showing the corresponding reporter combination. Black squares represent unidentifiable glomeruli. Rows were distributed as follows: Upper rows: neuroblast events, middle rows: GMC events and lower rows: no cascade progression. Colored vertical lines show the limits where the majority of transitions occurred, corresponding to the different heat-shock windows. All images are maximum intensity projections of confocal images. Green, red and gray, immunohistochemistry for V5 (YFP), RFP and Dpn respectively. None of the neuroblasts expressed CFP in any of these experiments. All brains were fixed at wandering larva stage. For simplicity, the fusion of Cas9 to a Geminin degron is not shown in these cartoons (see Fig 5). Scale bars = 100 μm. n= 48 (8-12h AEL), 30 (36-48h ALH), 25 (48-60h ALH) and 24 (double) brains. In the schemes, circled numbers represent the order of events occurring in the cascade.

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Supplementary Note, Supplementary Tables 1 and 2, and plasmid sequences.

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Garcia-Marques, J., Espinosa-Medina, I., Ku, K. et al. A programmable sequence of reporters for lineage analysis. Nat Neurosci (2020). https://doi.org/10.1038/s41593-020-0676-9

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