Biology subjects

Migliori, V.

Publications and source records attributed to Migliori, V..

4 recordsLinked to original sources

bioRxiv2026

STEER-seq: a recombinase based platform for pooled multimodal single-cell perturbation screening

Pooled gain-of-function screening is a powerful method for screening for transcription factors whose overexpression leads to changes in cellular identity. However, variable transgene copy number, integration site and genomic context from random viral integration can confound quantitative comparisons between perturbations. We present STEER-seq (Single-cell Transgene Expression via Exchange Recombination sequencing), a virus-free platform for pooled ORF gain-of-function screening in human induced pluripotent stem cells (hiPSCs). STEER-seq combines irreversible Bxb1-mediated integration at a defined genomic safe-harbour locus with promoterless donor libraries and barcode-enabled perturbation tracking, enabling single-copy expression from a common promoter and genomic context and direct recovery of perturbation identity by single-cell sequencing. In a focused haematopoietic screen, SPI1 expression alone was sufficient to drive differentiation towards HSC/MPP-like progenitors in the absence of exogenous cytokines, with transient induction generating progenitors that retained multilineage haematopoietic differentiation potential. Joint RNA-chromatin accessibility profiling linked transcription factor-induced transcriptional responses to chromatin remodelling and enabled reconstruction of perturbation-induced gene regulatory networks, including a RUNX2-driven osteoblast programme. Scaling STEER-seq to around 2,000 human coding sequences enabled systematic discovery of transcription factors capable of inducing distinct cell-state programmes, including neural progenitor-, melanocyte- and vascular smooth muscle-like states, as well as maintenance of pluripotency. STEER-seq provides a scalable, quantitative framework for single-cell and multimodal gain-of-function screening, enabling functional discovery and engineering of human cell identity.

genomics↗

bioRxiv2024

ONE-STEP tagging: a versatile method for rapid site-specificintegration by simultaneous reagent delivery

We present a novel, versatile genome editing method termed ONE-STEP tagging, which combines CRISPR-Cas9-mediated targeting with Bxb1 integrase-based site-specific integration for efficient, precise, and scalable protein tagging. Applied in hiPSCs, cancer cells, and primary T cells, this system enables rapid generation of endogenously tagged cell lines. By enhancing the nuclear localization signal (NLS) of the catalytically superior eeBxb1 integrase and co-delivering a DNA-PK inhibitor (AZD-7648), we achieved up to [~]90% integration efficiency at the ACTR10 locus. ONE-STEP tagging is robust across diverse loci and cell types and supports large DNA cargo integration, with efficiencies reaching 16.6% for a 14.4 kb construct. The method also enables multiplexed tagging of multiple proteins within the same cell and allows simultaneous CRISPR-based editing at secondary loci, such as gene knockouts or homology-directed repair (HDR) insertions. Importantly, we demonstrate successful application in primary T cells by targeting the T cell receptor (TCR) locus while simultaneously knocking out B2M, a key step toward generating immune-evasive, off-the-shelf CAR-T cells. Additionally, we introduce a dual-cassette version of the method compatible with universal donor plasmids, allowing use of entirely off-the-shelf reagents. Together, these advances establish ONE-STEP tagging as a powerful tool for both basic and therapeutic genome engineering.

molecular biology↗

bioRxiv2023

Pseudouridine guides germline small RNA transport and epigenetic inheritance

Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes1. Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation2,3. In C. elegans, these inherited small RNAs have poly (UG) tails4, but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine ({Psi}) is the most abundant RNA modification but has not been explored in small RNAs. Here, we develop novel assays to detect {Psi} in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs and their precursors. We also detect substantial enrichment in germline small RNAs, namely epigenetically activated siRNAs (easiRNAs) in Arabidopsis pollen, and piwi-interacting piRNAs in mouse testis. In pollen, pseudouridylated easiRNAs are localized to sperm cells, and we found that PAUSED/HEN5 (PSD), the plant homolog of Exportin-t, interacts genetically with {Psi} and is required for transport of easiRNAs into sperm cells from the vegetative nucleus. We further show that Exportin-t is required for the triploid block: chromosome dosage-dependent seed lethality that is epigenetically inherited from pollen. Thus, {Psi} has a conserved role in marking inherited small RNAs in the germline. One-Sentence SummaryPseudouridine marks germline small RNAs in plants and mammals, impacting epigenetic inheritance via nuclear transport.

genetics↗

bioRxiv2019

RNA modifications detection by comparative Nanopore direct RNA sequencing

RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. To date, over 150 naturally occurring PTMs have been identified, however the overwhelming majority of their functions remain elusive. In recent years, a small number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing (DRS) technology has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework to evaluate the presence of modifications in DRS data. To do so, we compare an RNA sample of interest against a non-modified control sample. Our strategy does not require a training set and allows the use of replicates to model biological variability. Here, we demonstrate the ability of Nanocompore to detect RNA modifications at single-molecule resolution in human polyA+ RNAs, as well as in targeted non-coding RNAs. Our results correlate well with orthogonal methods, confirm previous observations on the distribution of N6-methyladenosine sites and provide novel insights into the distribution of RNA modifications in the coding and non-coding transcriptomes. The latest version of Nanocompore can be obtained at https://github.com/tleonardi/nanocompore.

genomics↗