Biology subjects

Borrajo, J.

Publications and source records attributed to Borrajo, J..

4 recordsLinked to original sources

bioRxiv2024

Live-cell transcriptomics with engineered virus-like particles

The transcriptional state of a cell provides a multi-parameter representation of gene expression programs that reflect its identity and phenotype. However, current transcriptomic profiling technologies result in destruction of the biological sample, preventing direct analysis of transcriptional dynamics in the same living cells over time. Here, we developed a synthetic RNA export system called cellular self-reporting to address this fundamental technological limitation. We repurposed the murine leukemia virus retroviral protein Gag to enable diverse types of immortalized and primary mammalian cells to package cellular RNA molecules in virus-like particles (VLPs) for export into the extracellular environment. We applied self-reporting to interrogate the transcriptome-wide dynamics that occur during neuronal differentiation from induced pluripotent stem cells and detected gene expression profiles from individual live cells. Leveraging this genetically encodable approach, we expanded the capabilities of self-reporting through molecular engineering of VLP components. Pseudotyping VLPs with epitope-tagged envelope proteins enabled multiplexed selective live-cell readout of transcriptional states from heterogeneous co-cultures. Furthermore, structure-guided protein engineering of Gag fusions with human RNA binding domains improved the mRNA representation in self-reporting readouts and enabled the directed export of libraries of synthetic barcode transcripts. Taken together, this work establishes self-reporting as a facile and broadly enabling technology for live-cell, transcriptome-scale profiling of dynamic processes across diverse cell types and biological applications.

bioengineering↗

bioRxiv2024

Pharmacological SOS1 inhibitor BI-3406 demonstrates in vivo anti-tumor activity comparable to SOS1 genetic ablation in KRAS mutant tumors

Resistance to KRASmut inhibitors frequently arises, warranting further searches for anti-RAS cancer therapies. We evaluated the tolerability and efficacy of SOS1 pharmacological inhibition in comparison to genetic ablation in different KRAS-dependent tumor settings. Contrary to the rapid lethality caused by SOS1 genetic ablation in SOS2KO mice, SOS1 pharmacological inhibition by its specific inhibitor BI-3406 did not significantly affect animal weight/viability nor cause noteworthy systemic toxicity. In BI-3406-treated KRASmut MEFs, we observed significantly reduced RAS-GTP levels and RAS downstream signaling, as well as decreased tumor burden and slower disease progression resulting from tumor-intrinsic and extrinsic therapeutic drug effects. In vivo analyses of KRASG12D allografts in immunocompromised mice and KRASG12D-driven lung adenocarcinomas in immunocompetent mice showed that systemic BI-3406 treatment impaired tumor growth and downmodulated components of the tumor microenvironment comparably to the KRASG12D inhibitor MRTX1133. Markedly stronger synergistic antitumor effects were observed upon concomitant BI-3406+MRTX113 treatment, confirming SOS1 as an actionable therapy target in RAS-dependent cancers.

cancer biology↗

bioRxiv2023

Programmable multi-kilobase RNA editing using CRISPR-mediated trans-splicing

Current gene editing approaches in eukaryotic cells are limited to single base edits or small DNA insertions and deletions, and remain encumbered by unintended permanent effects and significant challenges in the delivery of large DNA cargo. Here we describe Splice Editing, a generalizable platform to correct gene transcripts in situ by programmable insertion or replacement of large RNA segments. By combining CRISPR-mediated RNA targeting with endogenous cellular RNA-splicing machinery, Splice Editing enables efficient, precise, and programmable large-scale editing of gene targets without DNA cleavage or mutagenesis. RNA sequencing and measurement of spliced protein products confirm that Splice Editing achieves efficient and specific targeted RNA and protein correction. We show that Splice Editors based on novel miniature RNA-targeting CRISPR-Cas systems discovered and characterized in this work can be packaged for effective delivery to human cells and affect different types of edits across multiple targets and cell lines. By editing thousands of bases simultaneously in a single reversible step, Splice Editing could expand the treatable disease population for monogenic diseases with large allelic diversity without the permanent unintended effects of DNA editing. One-sentence summaryCRISPR-guided trans-splicing enables efficient and specific recombination of large RNA molecules in mammalian cells, with broad applications in therapeutic development for genetic diseases and as a research tool for the study of basic RNA biology.

genomics↗

bioRxiv2020

LAMP-Seq: Population-Scale COVID-19 Diagnostics Using a Compressed Barcode Space

The ongoing SARS-CoV-2 pandemic has already caused devastating losses. Exponential spread can be slowed by social distancing and population-wide isolation measures, but those place a tremendous burden on society, and, once lifted, exponential spread can re-emerge. Regular population-scale testing, combined with contact tracing and case isolation, should help break the cycle of transmission, but current detection strategies are not capable of such large-scale processing. Here we present a protocol for LAMP-Seq, a barcoded Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method that is highly scalable. Individual samples are stabilized, inactivated, and amplified in three isothermal heat steps, generating barcoded amplicons that can be pooled and analyzed en masse by sequencing. Using unique barcode combinations per sample from a compressed barcode space enables extensive pooling, potentially further reducing cost and simplifying logistics. We validated LAMP-Seq on 28 clinical samples, empirically optimized the protocol and barcode design, and performed initial safety evaluation. Relying on world-wide infrastructure for next-generation sequencing, and in the context of population-wide sample collection, LAMP-Seq could be scaled to analyze millions of samples per day.

molecular biology↗