Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters

Lalanne, Jean-Benoît; Regalado, Samuel G.; Domcke, Silvia; Calderon, Diego; Martin, Beth; Li, Tony; Suiter, Chase C.; Lee, Choli; Trapnell, Cole; Shendure, Jay

doi:10.1101/2022.12.10.519236

Cited by 14 publications

(19 citation statements)

References 111 publications

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“…iPSC derived organoids are also a promising avenue for investigating brain development in non-human primates, including comparative studies. Another technological development that could be used to expand upon this study is single-cell MPRA ( 86 , 87 ). While currently limited to a small number of sequences, this approach could eventually overcome some limitations we faced testing cell-type specific DAs in a bulk assay.…”

Section: Discussionmentioning

confidence: 99%

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex

Deng

Whalen

Steyert

et al. 2023

Preprint

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Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

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Section: Discussionmentioning

confidence: 99%

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex

Deng

Whalen

Steyert

et al. 2023

Preprint

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“…The piggyFlex vector has both antibiotic (puromycin) and fluorophore (GFP) markers, enabling flexibly stringent selection for cells with higher numbers of gRNA integrants. Additionally, this vector design allows for gRNA transcript capture during single-cell library preparation 18 ( Fig. S1C ).…”

Section: Resultsmentioning

confidence: 99%

“…b) PiggyFlex gRNA library contents by target category. c) PiggyFlex construct design.PiggyFlex is a piggyBac transposon-based gRNA delivery vector equipped with a dual antibiotic (puromycin) and fluorophore (GFP) selection cassette that enables enrichment for cells with many integrated gRNAs18 . PiggyFlex enables direct capture of gRNA transcripts or optional capture of gRNA-associated barcodes from GFP mRNA via CS2 or polydT capture.…”

mentioning

confidence: 99%

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

Chardon

McDiarmid

Page

et al. 2023

Preprint

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CRISPR-based gene activation (CRISPRa) is a promising therapeutic approach for gene therapy, upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically and potently upregulating target genes, including autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate therapeutically relevant genes in a cell type-specific manner.

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“…transient TF activation 132,133 ), and intercellular expression variance 134 . Stably integrated translated reporters 135 and single cell MPRAs 136,137 theoretically overcome many of these limitations, but have so far not been widely applied at scale. While reporter assays can produce rich data, it will also be useful to measure the biochemical intermediates between sequence and expression, for instance detecting TF binding using enzymatic footprinting 138 .…”

Section: Current and Needed Technologies To Facilitate Large-scale Sy...mentioning

confidence: 99%

Hold out the genome: A roadmap to solving the cis-regulatory code

Boer

Taipale

2023

Preprint

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Gene expression is regulated by transcription factors that work together to read cis-regulatory DNA sequences. The “cis-regulatory code” - the rules that cells use to determine when, where, and how much genes should be expressed - has proven to be exceedingly complex, but recent advances in the scale and resolution of functional genomics assays and Machine Learning have enabled significant progress towards deciphering this code. However, we will likely never solve the cis-regulatory code if we restrict ourselves to models trained only on genomic sequences; regions of homology can easily lead to overestimation of predictive performance, and there is insufficient sequence diversity in our genomes to learn all relevant parameters. Fortunately, randomly synthesized DNA sequences enable us to test a far larger sequence space than exists in our genomes in each experiment, and designed DNA sequences enable a targeted query of the sequence space to maximally improve the models. Since cells use the same biochemical principles to interpret DNA regardless of its source, models that are trained on these synthetic data can predict genomic activity, often better than genome-trained models. Here, we provide an outlook on the field, and propose a roadmap towards solving the cis-regulatory code by training models exclusively on non-genomic DNA sequences, and using genomic sequences solely for evaluating the resulting models.

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Multiplex profiling of developmental enhancers with quantitative, single-cell expression reporters

Cited by 14 publications

References 111 publications

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

Hold out the genome: A roadmap to solving the cis-regulatory code

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