Biology subjects

Cubitt, L.

Publications and source records attributed to Cubitt, L..

3 recordsLinked to original sources

bioRxiv2025

PETRA: Prime editing of transcribed regulatory elements to assay expression

Predicting how changes in human DNA sequence impact gene expression remains challenging. Here, we present PETRA (Prime Editing of Transcribed Regulatory elements to Assay expression), a multiplexed genome editing method to quantify the effects of regulatory variants at scale. PETRA leverages the delivery of variants to abundantly transcribed regions of genes such that sequence-specific effects on mRNA expression can be read out by amplicon sequencing. We demonstrate PETRA in Jurkat cells by scoring 13,935 six-nucleotide insertions delivered to the 5 untranslated regions (5 UTRs) of four genes important for T cell responses, namely VAV1, IL2RA, CD28 and OTUD7B. Effects on expression are linked to the creation of new transcription factor binding sites (TFBSs), as well as to alterations in splicing and translation initiation. Combinatorial delivery of TFBSs identified using PETRA generates alleles that increase mRNA expression more than 10-fold. Additionally, we extend PETRA to primary human T cells to compare effects across cell types and use our data to assess the performance of computational models. These results establish PETRA as a flexible means of dissecting and reprogramming the logic of gene regulation across genomic contexts and cell types.

genomics↗

bioRxiv2024

RNA editing is a molecular clock in unmodified human cells

Despite major advances in spatial RNA sequencing, the ability to extract temporal information in RNA sequencing experiments is still limited. Here, we describe Transcriptome Timestamping (T2), a system which harnesses naturally occurring A-to-I editing of RNA transcripts in unmodified human cells to infer transcriptional history. T2 provides age estimates for individual RNA transcripts, and serves as an endogenous molecular recorder, differentiating between complex transcriptional programs. We show that T2 can identify transient and transitional transcriptional programs in primary differentiating monocytes that are not apparent from gene expression analysis alone, including a regulatory module in the monocyte-to-macrophage transition that, to our knowledge, has not yet been described in humans. Finally, we show that T2 can also be applied to single cell data, allowing us to identify transcriptional programs in heterogeneous populations, such as asynchronously dividing cells. T2 is a scalable approach to temporal transcriptomics that can be applied to track the activity of thousands of genes in unmodified, primary human cells and tissues, with no genetic engineering.

genomics↗

bioRxiv2017

A practical solution for preserving single cells for RNA sequencing

The design and implementation of single-cell experiments is often limited by their requirement for fresh starting material. We have adapted a method for histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP), or Lomants Reagent, to stabilise cell samples for single-cell transcriptomic applications. DSP is a reversible cross-linker of free amine groups that has previously been shown to preserve tissue integrity for histology while maintaining RNA integrity and yield in bulk RNA extractions. Although RNA-seq data from DSP-fixed single cells appears to be prone to characteristic artefacts, such as slightly reduced yield of cDNA and a detectable 3 bias in comparison with fresh cells, cell preservation using DSP does not appear to substantially reduce RNA complexity at the gene level. In addition, there is evidence that instantaneous fixation of cells can reduce inter-cell technical variability. The ability of DSP-fixed cells to retain commonly used dyes, such as propidium iodide, enables the tracking of experimental sub-populations and the recording of cell viability at the point of fixation. Preserving cells using DSP will remove several barriers in the staging of single-cell experiments, including the transport of samples and the scheduling of shared equipment for downstream single-cell isolation and processing.

genomics↗