{"database":"GEO","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Other":["ftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE330nnn/GSE330222/"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"omics_type":["Transcriptomics"],"species":["Homo sapiens"],"gds_type":["Expression profiling by high throughput sequencing"],"full_dataset_link":["https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE330222"],"repository":["GEO"],"entry_type":["GSE"],"additional_accession":[]},"is_claimable":false,"name":"Short-read and long-read single-cell sequencing capture distinct perturbation effects in CRISPR screens","description":"Background: Single-cell CRISPR screens have transformed functional genomics by enabling scalable, systematic investigation of gene function. Despite this, transcriptional complexity has largely been overlooked, with studies focusing on gene-level effects rather than isoforms. 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. Results: We compare two single-cell library preparation methods (5' 10x Genomics and Parse Biosciences Evercode) across Illumina short-read, Oxford Nanopore Technologies, and PacBio long-read sequencing, applying CRISPRi to silence three genes with distinct regulatory roles (DDX6, GEMIN5 and GFI1B) in K562 cells. While short-read methods can detect some alternative splicing events, only long-read sequencing consistently captures isoform-level changes. Although Parse Evercode provided even coverage across transcripts, we observed strong enrichment of intronic reads, limiting its utility for splicing analysis. The primary constraint of long-read approaches was depth: approximately 21 million reads are required for 80% saturation of splicing events in a single perturbation, underscoring the need for higher-throughput methods. Despite these limitations, we show that GEMIN5 perturbation produced modest differential expression but the most extensive splicing changes, an effect invisible to gene-level analysis, highlighting the importance of extending CRISPR screens to isoform-level readouts. Conclusions: We provide a practical framework for isoform-level analysis in single-cell CRISPR screens, identifying both the capabilities and current limitations of available approaches. As perturbation studies scale, long-read sequencing will be essential for comprehensive functional interpretation, capturing biology missed by gene-level analysis.","dates":{"publication":"2026/05/19"},"accession":"GSE330222","cross_references":{"GSM":["GSM9721529","GSM9721527","GSM9721528","GSM9721534","GSM9721532","GSM9721533","GSM9721530","GSM9721531"],"GPL":["26167","34284","34382"],"GSE":["330222"],"taxon":["Homo sapiens"]}}