CRISPR screens have been used to connect genetic perturbations with changes in gene expression and phenotypes. Here we describe a CRISPR-based, single-cell combinatorial indexing assay for transposase-accessible chromatin (CRISPR–sciATAC) to link genetic perturbations to genome-wide chromatin accessibility in a large number of cells. In human myelogenous leukemia cells, we apply CRISPR–sciATAC to target 105 chromatin-related genes, generating chromatin accessibility data for ~30,000 single cells. We correlate the loss of specific chromatin remodelers with changes in accessibility globally and at the binding sites of individual transcription factors (TFs). For example, we show that loss of the H3K27 methyltransferase EZH2 increases accessibility at heterochromatic regions involved in embryonic development and triggers expression of genes in the HOXA and HOXD clusters. At a subset of regulatory sites, we also analyze changes in nucleosome spacing following the loss of chromatin remodelers. CRISPR–sciATAC is a high-throughput, single-cell method for studying the effect of genetic perturbations on chromatin in normal and disease states.
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The scripts and pipeline for the analysis can be found at https://gitlab.com/sanjanalab/crispr-sciatac.
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We thank the entire Sanjana laboratory for support and advice. We thank J. Morris for help with eQTL resources, M. Zaran and R. Satija for computational resources and the NYGC Sequencing Platform and NYU Biology Genomics Core for sequencing resources. BL21(DE3) cells transformed with pET-PfuX7 were kindly provided by J. Gregory. N.L.-B. is supported by a postdoctoral fellowship from the Human Frontier Science Program Organization (no. LT000672/2019-L), an EMBO long-term fellowship (no. ALTF 826-2018) and the Weizmann Institute of Science National Postdoctoral Award Program for Advancing Women in Science. N.E.S. is supported by NYU and NYGC startup funds, NIH/NHGRI (nos. R00HG008171 and DP2HG010099), NIH/NCI (no. R01CA218668), DARPA (no. D18AP00053), the Sidney Kimmel Foundation, the Melanoma Research Alliance and the Brain and Behavior Foundation.
The New York Genome Center and New York University have applied for patents relating to the work in this article. N.E.S. is an adviser to Vertex.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–14 and Sequences.
Number of cells in each step of the CRISPR–sciATAC protocol.
Sequences of oligonucleotides for CRISPR–sciATAC, CRISPR libraries and RT–qPCR.
Gene and gRNA enrichment from essentiality screen.
ENCODE ChIP data sources.
Histone mark differential accessibility.
GO enrichment results for differential accessibility in EZH2-targeted cells.
Transcription factor binding site differential accessibility.
Cost comparison between CRISPR–sciATAC and Perturb–ATAC protocols.
Time comparison between CRISPR–sciATAC and Perturb–ATAC protocols.
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Liscovitch-Brauer, N., Montalbano, A., Deng, J. et al. Profiling the genetic determinants of chromatin accessibility with scalable single-cell CRISPR screens. Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-00902-x