Lundqvist, S. | bioRxiv Science

bioRxiv2026

Isoform-level resolution in single-cell CRISPR screens reveals hidden functional consequences of gene perturbation

Single-cell CRISPR screens have enabled systematic investigation of gene function, but studies have largely focused on gene-level effects, overlooking transcriptional complexity and isoform usage. Methods capable of capturing splicing and isoform usage have emerged, including long-read sequencing and alternative library preparation strategies, but their suitability for large-scale perturbation screens remains unevaluated. We compare two library preparation methods (10x Genomics and Parse Biosciences) across Illumina short-read, Oxford Nanopore, and PacBio long-read sequencing, applying CRISPRi to silence three genes with distinct regulatory roles (DDX6, GEMIN5, GFI1B) in K562 cells. While short-read methods detected some splicing events, only long-read sequencing consistently captured isoform-level changes. Although Parse provided even transcript coverage, we observed strong intronic read enrichment, limiting its utility for splicing analysis. The primary constraint of long-read approaches was sequencing depth: [~]21 million reads are needed for 80% saturation of splicing events in a single perturbation. Notably, GEMIN5 knockdown produced only modest differential expression but the most extensive splicing changes, an effect invisible to gene-level analysis, underscoring the value of isoform-level screens. We provide a practical framework for isoform-level analysis in single-cell CRISPR screens, identifying current capabilities and limitations. As perturbation studies scale, long-read sequencing will be essential for comprehensive functional interpretation, capturing biology missed by gene-level analysis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=49 SRC="FIGDIR/small/737410v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@14bb963org.highwire.dtl.DTLVardef@783205org.highwire.dtl.DTLVardef@11c0233org.highwire.dtl.DTLVardef@1d468fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

DiMeLo-cito: a one-tube protocol for mapping protein-DNA interactions reveals CTCF bookmarking in mitosis

Genome regulation relies on complex and dynamic interactions between DNA and proteins. Recently, powerful methods have emerged that leverage third-generation sequencing to map protein-DNA interactions genome-wide. For example, Directed Methylation with Long-read sequencing (DiMeLo-seq) enables mapping of protein-DNA interactions along long, single chromatin fibers, including in highly repetitive genomic regions. However, DiMeLo-seq involves lossy centrifugation-based wash steps that limit its applicability to many sample types. To address this, we developed DiMeLo-cito, a single-tube, wash-free protocol that maximizes the yield and quality of genomic DNA obtained for long-read sequencing. This protocol enables the interrogation of genome-wide protein binding with as few as 100,000 cells and without the requirement of a nuclear envelope, enabling confident measurement of protein-DNA interactions during mitosis. Using this protocol, we detected strong binding of CTCF to mitotic chromosomes in diploid human cells, in contrast with earlier studies in karyotypically unstable cancer cell lines, suggesting that CTCF "bookmarks" specific sites critical for maintaining genome architecture across cell divisions. By expanding the capabilities of DiMeLo-seq to a broader range of sample types, DiMeLo-cito can provide new insights into genome regulation and organization.

genomics↗