Summary Spatial genome organization and its effect on transcription remains a fundamental question. We applied an advanced ChIA-PET strategy to comprehensively map higher-order chromosome folding and specific chromatin interactions mediated by CTCF and RNAPII with haplotype specificity and nucleotide resolution in different human cell lineages. We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation, whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes towards CTCF-foci for coordinated transcription. Furthermore, we show that haplotype-variants and allelic-interactions have differential effects on chromosome configuration influencing gene expression and may provide mechanistic insights into functions associated with disease susceptibility. 3D-genome simulation suggests a model of chromatin folding around chromosomal axes, where CTCF is involved in defining the interface between condensed and open compartments for structural regulation. Our 3D-genome strategy thus provides unique insights in the topological mechanism of human variations and diseases.

Genomes of higher organisms are extensively folded into three-dimensional (3D) chromosome territories within the nucleus 1. Advanced 3D genome mapping methods that combine proximity ligation and high-throughput sequencing (Hi-C) 2 , plus chromatin immunoprecipitation (ChIA-PET) 3 , have revealed topologically associating domains (TADs) 4 with frequent chromatin contacts and have identified chromatin loops mediated by specific protein factors for insulation and transcriptional regulation 5-7. However, these methods rely on pairwise proximity ligation and reflect population-level views, and thus cannot reveal the detailed nature of chromatin interactions. Although single-cell Hi-C 8 could potentially overcome this issue, it may be limited by data sparsity inherent to current single-cell assays. Recent advances in microfluidics have opened new opportunities for droplet-based genomic analysis 9 , yet this approach has not been adapted to chromatin interaction analysis. Here, we describe a strategy for multiplex chromatin interaction analysis via droplet-based and barcode-linked sequencing (ChIA-Drop). We demonstrate the robustness of ChIA-Drop in capturing complex chromatin interactions with unprecedented single-Reprints and permissions information is available at www.nature.com/reprints.

Summary Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a robust method to capture genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution for detecting chromatin interactions, whereas 3C/Hi-C approaches could not achieve. The original ChIA-PET protocol generates short paired-end tags (2×20 bp) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end-tags is increased (up to 2×250 bp). The longer PET reads not only improve the tag mapping efficiency but also increase the probability of covering phased SNPs, which allows haplotype-specific chromatin interactions identification. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with bridge linker, Tn5 tagmentation, PCR amplification, and high-throughput sequencing. To a well-trained molecular biologist, it typically takes six days from cell harvesting to the completion of library construction, up to a further 36 hours for DNA sequencing, and less than 20 hours for processing raw sequencing reads.

Alternative splicing allows expression of mRNA isoforms from a single gene, expanding the diversity of the proteome. Its prevalence in normal biological and disease processes warrant precise tools for modulation. Here we report the engineering of CRISPR Artificial Splicing Factors (CASFx) based on RNA-targeting CRISPR-Cas systems. We show that simultaneous exon inclusion and exclusion can be induced at distinct targets by differential positioning of CASFx. We also create inducible CASFx (iCASFx) using the FKBP-FRB chemical-inducible dimerization domain, allowing small molecule control of alternative splicing. Finally, we demonstrate the activation of SMN2 exon 7 splicing in spinal muscular atrophy (SMA) patient fibroblasts, suggesting a potential application of the CASFx system.