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Cited by 118Genomics and Chromatin DynamicsSingle-cell and spatial transcriptomicsCRISPR and Genetic Engineering

A scalable platform for the development of cell-type-specific viral drivers

Open access
Hrvatin, Sinisa, Tzeng, Christopher P, Nagy, M Aurel, Stroud, Hume, Koutsioumpa, Charalampia, Wilcox, Oren F, Assad, Elena G, Green, Jonathan +3 more·2019·eLife
DOI 10.7554/elife.48089PMC PMC6776442
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Abstract

Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression. Recent studies characterizing individual enhancers have revealed their potential to direct heterologous gene expression in a highly cell-type-specific manner. However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism. We have developed PESCA, a scalable and generalizable method that leverages ATAC- and single-cell RNA-sequencing protocols, to characterize cell-type-specific enhancers that should enable genetic access and perturbation of gene function across mammalian cell types. Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity. This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.

AI snapshot

Written from the paper’s full text.

TL;DR

The authors developed PESCA, a scalable platform combining ATAC-seq and single-nucleus RNA-seq to screen hundreds of gene regulatory elements in vivo for cell-type-specific viral expression. Applying PESCA to mouse visual cortex, they identified AAV drivers that specifically target somatostatin-expressing interneurons and showed these reagents can modulate neuronal activity.

Key findings

  • PESCA screened 287 conserved SST-enriched GREs as 861 GRE-barcode pairs in AAV9; 32,335 nuclei were analyzed across two mice, with PCR enrichment increasing viral transcript recovery 382-fold to an average of 15.6 unique viral transcripts per nucleus.
  • Several GREs drove SST-specific expression; the top five hits were GRE12, GRE19, GRE22, GRE44, and GRE80. In situ validation of GRE12, GRE22, and GRE44 showed 90.7 ± 2.1%, 72.9 ± 4.2%, and 95.8 ± 0.6% of GFP+ cells were SST+, respectively, versus 27.2 ± 1.9% for a ΔGRE control.
  • GRE12, GRE22, and GRE44 also reduced GFP expression in SST− cells by 3–32-fold and showed low overlap with PV+ or VIP+ cells (e.g., 1.4–4.3% PV+ and 1.2–1.7% VIP+ for the three GREs).
  • rAAV-GRE44-GFP+ SST neurons differed electrophysiologically from nearby GFP− SST neurons, including broader action potentials, lower rheobase, slower action potential rise, and lower maximal firing frequencies. rAAV-GRE12-Gq-DREADD-tdTomato enabled CNO-induced depolarization and increased firing in infected SST+ cells without significantly changing nearby uninfected pyramidal neurons.

Methods

  • Comparative ATAC-seq was performed on INTACT-purified SST, PV, and VIP nuclei from adult mouse cortex; peaks were called with HOMER and MACS2, filtered for mammalian conservation, and ranked by SST enrichment to select 287 GREs.
  • Each GRE was PCR-amplified, paired with three 10-mer barcodes, cloned into an AAV vector with a human beta-globin promoter driving GFP and a 3′UTR barcode, and packaged into AAV9. The pooled library was injected into V1 of two 6-week-old C57BL/6 mice, and nuclei were processed with inDrops snRNA-seq.
  • Viral transcripts were PCR-enriched, barcodes deconvolved by cell, nuclei clustered into ten cell types, and SST enrichment computed as mean expression in Sst+ versus Sst− cells. Hits were validated by fluorescence imaging in Sst-Cre; Ai14 and Vip-Cre; Ai14 mice, immunostaining for PV, whole-cell electrophysiology, and DREADD-mediated activity modulation.

Limitations

  • The paper states that further work is needed to identify which specific molecular subtypes of SST interneurons are targeted, and that GRE44 expression could not be confirmed as restricted to a specific transcriptionally defined SST subtype.
  • The paper estimates sufficient power to assess approximately 2/3 of the 287 GREs at the reported sequencing depth; less abundant GRE-driven transcripts may not allow reliable evaluation of cell-type specificity.
  • The paper notes that snRNA-seq specificity and protein-level specificity differed for GRE12, GRE22, and GRE44, which could be attributed to multiple factors. Evident from the text: the screen and validation are limited to mouse V1 and mainly SST/PV/VIP cortical interneurons, with only three top GREs validated in situ.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.7554/elife.48089

Cites

46 of 46 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 20 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The control rAAV-ΔGRE-GFP showed a strikingly different pattern of GFP expression concentrated around the sites of injection, with expression in a larger number of cells.

    “By contrast, the control rAAV-ΔGRE-GFP showed a strikingly different pattern of GFP expression concentrated around the sites of injection, with expression in a larger number of cells (Figure 3d).”
    Quote in the paperSubstantial claim
  • Findingown data

    Among the 287 GREs tested, several viral drivers were identified that promoted highly specific reporter expression in the Sst+ subpopulation (q < 0.01, fold-change >7).

    “Among the 287 GREs tested, several viral drivers were identified that promoted highly specific reporter expression in the Sst+ subpopulation (q < 0.01, fold-change >7, Figure 2h–j, Figure 2—figure supplement 5c–e).”
    Quote in the paperSubstantial claim
  • Findingown data

    Fluorescent signal analysis indicated the percentage of GFP+ cells that were either VIP+ or PV+ (rAAV-SST12-GFP+ [2.6 ± 2.6%], rAAV-GRE22-GFP+ [3.5 ± 2.0%] and rAAV-GRE44-GFP+ [6.0 ± 2.7%]).

    “Fluorescent signal analysis indicated the percentage of GFP+ cells that were either VIP+ or PV+ (rAAV-SST12-GFP+ [2.6 ± 2.6%], rAAV-GRE22-GFP+ [3.5 ± 2.0%] and rAAV-GRE44-GFP+ [6.0 ± 2.7%], Figure 3h).”
    Quote in the paperSubstantial claim
  • Findingown data

    All tdTomato+ cells showed striking sensitivity to CNO.

    “All tdTomato+ cells showed striking sensitivity to CNO, as indicated by significantly increased firing rates in response to depolarizing current steps and depolarized resting membrane potentials (Figure 4e–g).”
    Quote in the paperSubstantial claim
  • Findingown data

    ATAC-Seq identified genomic regions with enhanced accessibility (peaks) in SST (n = 57,932), PV (n = 61,108), and VIP (n = 79,124) chromatin samples.

    “The assay for transposase-accessible chromatin using sequencing (ATAC-Seq) (Buenrostro et al., 2015), which identifies nucleosome-depleted gene regulatory regions, was then used to identify genomic regions with enhanced accessibility (i.e., peaks) in the SST (n = 57,932), PV (n = 61,108), and VIP (n = 79,124) chromatin samples (Figure 1b,c, Figure 1—figure supplement 1, Materials and methods).”
    Quote in the paperSubstantial claim
  • May combine two claims. 26
  • Classified as probably not a substantial claim. 26

  • May combine two claims.

    Findingown data

    The majority of rAAV-GRE12-GFP+ and rAAV-GRE44-GFP+ SST+ cells were found to reside in layers IV and V, which was distinct from the distribution observed for the full SST+ cell population in visual cortex (p=1.3 × 10−6, p<2.2 × 10−16, respectively).

    “Intriguingly, the majority of rAAV-GRE12-GFP+ and rAAV-GRE44-GFP+ SST+ cells were found to reside in layers IV and V, which was distinct from the distribution observed for the full SST+ cell population in visual cortex (p=1.3 × 10−6, p<2.2 × 10−16, respectively, Mann-Whitney U test, two-tailed, Figure 3i, Figure 3—figure supplement 5).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingown data

    The remaining 36,215 genomic regions were ranked by an enrichment of ATAC-seq signal in the SST samples over PV/VIP, and the top 287 most enriched GREs were selected for functional screening.

    “The remaining 36,215 genomic regions were ranked by an enrichment of ATAC-seq signal in the SST samples over PV/VIP (Materials and methods), and the top 287 most enriched GREs were selected for functional screening to identify enhancers that drive gene expression selectively in SST interneurons of the primary visual cortex (Figure 1d, Supplementary file 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Many rAAV-GRE12/22/44-GFP virally infected cells were SST-positive: 90.7 ± 2.1% for rAAV-GRE12-GFP (170 cells, four animals); 72.9 ± 4.2% for rAAV-GRE22-GFP (1164 cells, three animals), and 95.8 ± 0.6% for rAAV-GRE44-GFP (759 cells, four animals).

    “Many rAAV-GRE12/22/44-GFP virally infected cells were SST-positive, as indicated by the high degree of overlapping GFP and tdTomato expression: 90.7 ± 2.1% for rAAV-GRE12-GFP (170 cells, four animals); 72.9 ± 4.2% for rAAV-GRE22-GFP (1164 cells, three animals), and 95.8 ± 0.6% for rAAV-GRE44-GFP (759 cells, four animals).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sets of three barcodes associated with the same GRE showed highly statistically correlated enrichment scores (r = 0.52 ± 0.05, p<2.2 × 10−16), which were significantly lower when barcodes were randomly shuffled (shuffled r = 0.002 ± 0.06; p=0.003).

    “As expected, sets of three barcodes associated with the same GRE showed highly statistically correlated enrichment scores (r = 0.52 ± 0.05, p<2.2 × 10−16) (Figure 2e,f, Figure 2—figure supplement 4), which were significantly lower when barcodes were randomly shuffled (shuffled r = 0.002 ± 0.06; Wilcox test between data and shuffled data, p=0.003).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PCR enrichment increased the viral transcript recovery 382-fold in the sampled nuclei, to an average of 15.6 unique viral transcripts, 6.0 unique GRE-barcodes, and 5.7 unique GREs per cell.

    “PCR enrichment increased the viral transcript recovery 382-fold in the sampled nuclei, to an average of 15.6 unique viral transcripts, 6.0 unique GRE-barcodes, and 5.7 unique GREs per cell (Figure 2b, Figure 2—figure supplement 2c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Applying PESCA to the mammalian cerebral cortex, we found enhancers and generated viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity.

    “Focusing on the highly heterogeneous mammalian cerebral cortex, we apply PESCA to find enhancers and generate viral reagents capable of accessing and manipulating a subset of somatostatin-expressing cortical interneurons with high specificity.”
    • Also stated in: § Introduction · plain
      “We validated the efficacy of PESCA in the murine primary visual cortex by identifying GREs that confine AAV expression to somatostatin (SST)-expressing interneurons and showed that these vectors can be used to modulate neuronal activity selectively in SST neurons.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression.
    • Rests on an assumption: The mammalian cerebral cortex is composed of over one hundred cell types, most of which cannot be individually accessed using existing tools.

  • May combine two claims.

    Findingown data

    The incorporation of GRE12, GRE22 and GRE44 into the rAAV both increased the number of SST+ GFP+ cells (1.7-2-fold) and dramatically (3-32-fold) decreased the number of SST- cells that expressed GFP.

    “Consistent with this hypothesis, the incorporation of GRE12, GRE22 and GRE44 into the rAAV both increased the number of SST+ GFP+ cells (1.7–2-fold) and dramatically (3–32-fold) decreased the number of SST- cells that expressed GFP (Figure 3g, Figure 3—figure supplement 3).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    The initial dataset contained 32,335 nuclei, with more than 200 unique non-viral transcripts (UMIs) assigned to each nucleus.

    “The initial dataset contained 32,335 nuclei, with more than 200 unique non-viral transcripts (UMIs) assigned to each nucleus.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The result was a list of 1164 10-base barcodes that fit the initial criteria.

    “The result was a list of 1164 10-base barcodes that fit our initial criteria.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    We recovered an average of 866 unique non-viral transcripts per nucleus, representing 610 unique genes.

    “We recovered an average of 866 unique non-viral transcripts per nucleus, representing 610 unique genes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Somatostatin-expressing cortical interneurons comprise several anatomically, electrophysiologically, and molecularly defined cell types whose dysfunction is associated with neuropsychiatric and neurological disorders.

    “for example, somatostatin-expressing cortical interneurons comprise several anatomically, electrophysiologically, and molecularly defined cell types whose dysfunction is associated with neuropsychiatric and neurological disorders (Jiang et al., 2015; Muñoz et al., 2017; Tasic et al., 2018).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Jiang et al., 2015 source

      Not checked in the free run.

    • cites Muñoz et al., 2017

      Not checked in the free run.

    • cites Tasic et al., 2018 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    rAAV-ΔGRE-GFP was expressed in SST+ cells as well as other neuronal subtypes across all layers, suggesting that increased labeling of rAAV-GRE12-GFP and rAAV-GRE44-GFP in layer IV and V was likely due to restricted gene expression and not restricted viral tropism.

    “By contrast, rAAV-ΔGRE-GFP was expressed in SST+ cells as well as other neuronal subtypes across all layers, suggesting that increased labeling of rAAV-GRE12-GFP and rAAV-GRE44-GFP in layer IV and V was likely due to restricted gene expression and not restricted viral tropism.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.

    “This study demonstrates the utility of this platform for developing new cell-type-specific viral reagents, with significant implications for both basic and translational research.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Each GRE was paired with three unique barcode sequences to minimize sequencing bias due to the choice of barcode sequence.

    “To minimize sequencing bias due to the choice of barcode sequence, each GRE was paired with three unique barcode sequences.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    GRE sequences did not alter viral tropism and GRE-driven vectors had broadly similar levels of expression.

    “suggesting that GRE sequences did not alter viral tropism and that GRE-driven vectors had broadly similar levels of expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The expression of sparsely detected GRE-driven transcripts may not be sufficient to allow evaluation of cell-type-specificity and increasing sequencing depth may allow screening and evaluation of a larger number of GREs.

    “These observations suggest that the expression of sparsely detected GRE-driven transcripts may not be sufficient to allow evaluation of cell-type-specificity and that by increasing sequencing depth we may be able to screen and evaluate a larger number of GREs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The GREs seemingly not only promote expression in SST+ cells but also greatly reduce background expression in SST- cells, indicating both enhancer and repressor functionality.

    “It is notable that the GREs seemingly not only promote expression in SST+ cells but also greatly reduce background expression in SST- cells, indicating both enhancer and repressor functionality.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    Functional evaluation of candidate GRE-driven viral vector expression across all cell types in the tissue of interest is currently laborious, expensive, and low-throughput, typically relying on the production of individual viral vectors and the assessment of expression across a limited number of cell types by in situ hybridization or immunofluorescence.

    “Moreover, functional evaluation of candidate GRE-driven viral vector expression across all cell types in the tissue of interest is currently laborious, expensive, and low-throughput, typically relying on the production of individual viral vectors and the assessment of expression across a limited number of cell types by in situ hybridization or immunofluorescence.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    We chose to focus on SST neurons because this population is known to be diverse and to be composed of several relatively rare subpopulations, and thus might serve as a good test case.

    “We chose to focus on SST neurons in the brain because this population is known to be diverse and to be composed of several relatively rare subpopulations (Muñoz et al., 2017; Tasic et al., 2018; Tasic et al., 2016), and thus might serve as a good test case.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Muñoz et al., 2017

      Not checked in the free run.

    • cites Tasic et al., 2018 source

      Not checked in the free run.

    • cites Tasic et al., 2016 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    Cell-type-restricted gene regulatory elements (GREs) have not yet been systematically identified.

    “Despite the potential of cell-type-specific enhancers to revolutionize neuroscience research, cell-type-restricted gene regulatory elements (GREs) have not yet been systematically identified.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Our findings highlight the utility of PESCA for identifying viral constructs that drive gene expression selectively in a subset of neurons and establish PESCA as a platform of broad interest to the research and gene therapy community, potentially enabling the generation of cell-type-specific AAVs for virtually any cell type.

    “As described below, our findings highlight the utility of PESCA for identifying viral constructs that drive gene expression selectively in a subset of neurons and establish PESCA as a platform of broad interest to the research and gene therapy community, potentially enabling the generation of cell-type-specific AAVs for virtually any cell type.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression.

  • May combine two claims.

    Interpretationown data

    Although the 27.2% overlap between rAAV-ΔGRE-GFP expression and SST+ cells suggests that our vector has some baseline preference for SST+ interneurons, the insertion of GRE12, GRE22 and GRE44 serves to effectively restrict AAV payload expression to SST+ interneurons.

    “Although the 27.2% overlap between rAAV-ΔGRE-GFP expression and SST+ cells suggests that our vector has some baseline preference for SST+ interneurons, the insertion of GRE12, GRE22 and GRE44 serves to effectively restrict AAV payload expression to SST+ interneurons.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    It has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism.

    “However, it has not yet been possible to systematically identify and test the function of enhancers for each of the many cell types in an organism.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The library was packaged into AAV9, which exhibits broad neural tropism and has previously been used to drive payload expression in cortical neurons.

    “The library was packaged into AAV9, which exhibits broad neural tropism and has previously been used to drive payload expression in cortical neurons (Cearley and Wolfe, 2006).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Cearley and Wolfe, 2006

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The PESCA platform represents a significant advancement over current approaches to viral vector design, as it enables the rapid in vivo screening of hundreds of GREs for enhanced cell-type-specificity without needing transgenic tools to evaluate their specificity.

    “This represents a significant advancement over current approaches to viral vector design, as it enables the rapid in vivo screening of hundreds of GREs for enhanced cell-type-specificity without needing transgenic tools to evaluate their specificity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based). May combine two claims.

    Interpretationreasoning

    Given the vast diversity of cell types in the brain, and the inability of our current tools to access most neuronal cell types, enhancer-driven viral reagents have the potential to become the next generation of cell-type-specific transgenic tools enabling facile, inexpensive, cross-species, and targeted observation and functional study of neuronal cell types and circuits.

    “Given the vast diversity of cell types in the brain, and the inability of our current tools to access most neuronal cell types, enhancer-driven viral reagents have the potential to become the next generation of cell-type-specific transgenic tools enabling facile, inexpensive, cross-species, and targeted observation and functional study of neuronal cell types and circuits.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression.
    • Rests on an assumption: The mammalian cerebral cortex is composed of over one hundred cell types, most of which cannot be individually accessed using existing tools.

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationreasoning

    If GRE12 effectively drives DREADD expression, the other GREs might be expected to as well.

    “and thus, if it effectively drives DREADD expression, the other GREs might be expected to as well.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    DREADDs are a commonly employed viral payload used to dynamically regulate neuronal activity in response to the synthetic ligand clozapine-N-oxide (CNO).

    “Designer receptors exclusively activated by designer drugs (DREADDs) are a commonly employed viral payload used to dynamically regulate neuronal activity in response to the synthetic ligand clozapine-N-oxide (CNO) (Armbruster et al., 2007).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Armbruster et al., 2007 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Series resistance was compensated by at least 60% in a voltage-clamp configuration before switching to current-clamp.

    “Series resistance was compensated by at least 60% in a voltage-clamp configuration before switching to current-clamp (‘I Clamp Normal’).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The highest mean background signal for SST, VIP and PV was conservatively chosen as the threshold for classifying GFP+ cells as SST+, VIP+ or PV+, respectively.

    “The highest mean background signal for SST, VIP and PV was conservatively chosen as the threshold for classifying GFP+ cells as SST+, VIP+ or PV+, respectively.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    The authors did not count nuclei prior to performing ATAC-seq because yields were low enough that counting would remove a large fraction of isolated nuclei and negatively impact the quality of the ATAC-seq experiment.

    “We did not count the nuclei prior to performing ATAC-seq, as yields were low enough that the process of counting would remove a large fraction of isolated nuclei and negatively impact the quality of the ATAC-seq experiment.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    An average of at least three trials for each current injection was calculated before and during CNO application.

    “An average of at least three trials for each current injection was calculated before and during CNO application.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductreasoning

    The PCR preferentially amplifies viral-derived transcripts while retaining the cell-barcode sequence necessary to assign each transcript to a particular cell/nucleus.

    “The result of the PCR is preferential amplification of the viral-derived transcripts, while simultaneously retaining the cell-barcode sequence necessary to assign each transcript to a particular cell/nucleus.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Viral-derived sequences were removed for the purposes of embedding clustering and cell type identification.

    “Viral-derived sequences were removed for the purposes of embedding clustering and cell type identification.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Single-cell screening approaches are not limited to GRE screening; PESCA can be easily adapted to assess the cell-type-specificity of viral capsid variants or other mutable aspects of viral design.

    “Moreover, single-cell screening approaches are not limited to GRE screening; PESCA can be easily adapted to assess the cell-type-specificity of viral capsid variants or other mutable aspects of viral design.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Animal experiments were approved by the National Institute Health and Harvard Medical School Institutional Animal Care and Use Committee, following ethical guidelines described in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.

    “Animal experiments were approved by the National Institute Health and Harvard Medical School Institutional Animal Care and Use Committee, following ethical guidelines described in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Membrane potentials in this study were not corrected for the liquid junction potential and are thus positively biased by 8 mV.

    “Membrane potentials in this study were not corrected for the liquid junction potential and are thus positively biased by 8 mV.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Viral barcode sequences were chosen to be at least three insertions, deletions, or substitutions apart from each other to minimize the effects of sequencing errors on the correct identification of each barcode.

    “Viral barcode sequences were chosen to be at least three insertions, deletions, or substitutions apart from each other to minimize the effects of sequencing errors on the correct identification of each barcode.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    50 colonies were Sanger sequenced to determine the correct pairing between GRE and barcode and the correct arrangement of the AAV vector.

    “50 colonies were Sanger sequenced to determine the correct pairing between GRE and barcode and the correct arrangement of the AAV vector.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Two independent peak calling algorithms were employed to ensure robust, reproducible peak calls.

    “Two independent peak calling algorithms were employed to ensure robust, reproducible peak calls.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The data were modeled and normalized using a negative binomial distribution, consistent with snRNA-seq experiments.

    “The data were modeled and normalized using a negative binomial distribution, consistent with snRNA-seq experiments.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Channels were imaged sequentially to avoid any optical crosstalk.

    “Channels were imaged sequentially to avoid any optical crosstalk.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationreasoning

    Given current evidence that the mechanisms of gene regulatory element function are conserved across tissues and species, it is likely that PESCA can be readily applied to other neuronal or non-neuronal cell types, diverse model organisms, tissues, and viral types.

    “Given current evidence that the mechanisms of gene regulatory element function are conserved across tissues and species, it is likely that PESCA can be readily applied to other neuronal or non-neuronal cell types, diverse model organisms, tissues, and viral types.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Mechanisms of gene regulatory element function are conserved across tissues and species.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Tiled V1 cortical areas of ~1.2 mm by ~0.5 mm were imaged at a single optical section to avoid counting the same cell across multiple optical sections.

    “Tiled V1 cortical areas of ~1.2 mm by ~0.5 mm were imaged at a single optical section to avoid counting the same cell across multiple optical sections.”
    Quote in the paperNot a substantial claim
44 of 44

  • The paper relies on this without support.

    Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression.

    “Enhancers are the primary DNA regulatory elements that confer cell type specificity of gene expression.”

  • The paper relies on this without support.

    The mammalian cerebral cortex is composed of over one hundred cell types, most of which cannot be individually accessed using existing tools.

    “For example, the mammalian cerebral cortex is composed of over one hundred cell types, most of which cannot be individually accessed using existing tools.”

  • The paper relies on this without support.

    Mechanisms of gene regulatory element function are conserved across tissues and species.

    “Given current evidence that the mechanisms of gene regulatory element function are conserved across tissues and species”

  • The paper relies on this without support.

    There is an urgent practical need for new tools to access, study, and manipulate specific cell types across complex tissues, organ systems, and animal models.

    “our study addresses the urgent practical need for new tools to access, study, and manipulate specific cell types across complex tissues, organ systems, and animal models”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Molecular classification of cell types enables genetic access to specific cell types using transgenic mice and enhancer-based viral vectors.

    “Finally, molecular classification of cell types enables genetic access to specific cell types using transgenic mice25–27 and, more recently, enhancer-based viral vectors28–32.”

    From A multimodal cell census and atlas of the mammalian primary motor cortex (2021) · cites this paper as [31]

  • Identifying cell type-specific enhancers can guide development of viral vectors and transgenic lines targeting cell types for functional studies.

    “Identifying cell type-specific enhancers can guide development of viral vectors and transgenic lines targeting cell types for functional studies (Graybuck et al., 2019; Hrvatin et al., 2019).”

    From Single-Cell Sequencing of Brain Cell Transcriptomes and Epigenomes (2021) · cites this paper as Hrvatin et al., 2019

  • Such data can serve as a basis for gene regulatory network analysis and for predicting cell-type-specific enhancers for viral targeting.

    “These data can be used as a starting point for analysis of gene regulatory networks, and for predicting cell type specific enhancers for use in viral targeting tools (Hrvatin et al., 2019; Mich et al., 2020).”

    From Single-Cell Sequencing of Brain Cell Transcriptomes and Epigenomes (2021) · cites this paper as Hrvatin et al., 2019

  • Short regulatory elements can restrict viral expression to broad neuronal classes in the cerebral cortex or to a subpopulation of a subtype.

    “We and others have identified short regulatory elements capable of restricting viral expression to broad neuronal classes within the cerebral cortex (i.e. pan-pyramidal5 and pan-interneuron6) or a select subpopulation of a particular subtype (i.e subset of Sst within layer five7).”

    From Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans (2020) · cites this paper as [7]

  • Single-cell transcriptomic and epigenetic technologies have accelerated systematic enhancer discovery.

    “systematic enhancer discovery has been accelerated by the recent development of technologies allowing for transcriptomic and epigenetic studies at single-cell resolution6–12.”

    From Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans (2020) · cites this paper as [7]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • PESCA library injection titers differ

    Minor

    The Results give the pooled library titer as 1 × 1013 viral genomes/mL, while the injection methods give 1.9 × 1013 genome copies/mL for the same experiment.

  • Number of barcodes is confused with number of GREs

    Minor

    The enrichment methods describe 287 barcodes, whereas the library construction states that 287 GREs were paired with three barcodes each, producing 861 barcodes.

  • Figure 3i labels all ΔGRE GFP-positive cells as SST-positive

    Minor

    Figure 3i calls the 2,066 ΔGRE GFP-positive cells GFP-positive/SST-positive, but Figure 3f says only 27.2% of ΔGRE GFP-positive cells are SST-positive. The stated count cannot describe the SST-positive subset of those cells.

3 of 3

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Related

Authors
Hrvatin, S., Tzeng, C. P., Nagy, M. A., Stroud, H., Koutsioumpa, C., Wilcox, O. F., Assad, E. G., Green, J., Harvey, C. D., Griffith, E. C., & Greenberg, M. E.
Affiliations
  • Department of Neurobiology, Harvard Medical School, Boston, United States
  • Harvard University (US)
Corresponding author
Greenberg, Michael E
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2019-09-23
Volume
8
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.48089
PMC
PMC6776442
OpenAlex
W2975034635
Semantic Scholar
c30c614cc04f16a6bb9ddeef94985e11003bb0c1
Cited by
118
References
46
Citations by year
2026: 20 · 2025: 23 · 2024: 16 · 2023: 19 · 2022: 12 · 2021: 14 · 2020: 12 · 2019: 2
FWCI
3.31
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institutes of Health (RF1MH11408101, 5T32AG000222-23, T32GM007753, MH107620, NS089521, NS108410)
  • Life Sciences Research Foundation (Vertex Fellowship)
  • Charles A. King Trust (Postdoctoral Fellowship)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords
cell type-specific enhancers, viral vectors, cerebral cortex, ATAC-seq, single-cell RNA sequencing, enhancer identification, cell type-specific gene expression, gene perturbation
MeSH terms
Animals, Cerebral Cortex, Gene Expression Regulation, Genes, Regulator, Genetic Vectors, Interneurons, Molecular Biology, Neurons, Neurophysiology, Recombinant Proteins, Somatostatin, Viruses, Animals, Genetically Modified, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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