Biology subjects

Ross-Thriepland, D.

Publications and source records attributed to Ross-Thriepland, D..

4 recordsLinked to original sources

bioRxiv2026

Comparative Single-Cell Profiling of CRISPR Knockout and Interference Defines Modality-Specific Strengths in Functional Genomics

Pooled CRISPR screens coupled with single-cell RNA sequencing enable high-throughput functional interrogation of gene regulatory networks, yet systematic comparisons of CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) remain limited. We established a single-cell CRISPRko workflow and benchmarked it against CRISPRi using 87 sgRNAs targeting 29 unfolded protein response genes. CRISPRko generated transcriptional phenotypes are highly concordant with CRISPRi, and induced comparable pathway-level responses. While CRISPRi allows direct assessment of target gene repression, CRISPRko provides an effective complementary approach for complete loss-of-function studies, expanding the toolkit for single-cell functional genomics.

genomics↗

bioRxiv2026

Scarless conditional sgRNAs via endogenous mascRNA processing enable rapid and temporally controlled genome editing

Precise temporal control of gene editing is essential for studying dynamic biological processes, interrogating essential gene function, and improving the interpretability of pooled perturbation screens. Cre-dependent single guide RNA (sgRNA) switches provide temporal regulation by coupling guide activation to site-specific recombination, but existing designs retain a loxP-derived 5' sequence (scar) on the mature sgRNA that can impair guide function. We developed a scarless conditional sgRNA platform that combines Cre-loxP recombination with endogenous RNA processing to restore the native sgRNA architecture following induction. A MALAT1-associated small cytoplasmic RNA (mascRNA) module was positioned upstream of the guide sequence such that, after Cre-mediated recombination, cellular RNase P and RNase Z remove the residual loxP-derived overhang, generating a mature sgRNA with an authentic 5' terminus. Using guides targeting endogenous cell-surface marker genes, the scarless design maintained stringent OFF-state control while improving ON-state editing performance compared with a conventional Cre-activated sgRNA switch, resulting in faster editing kinetics, greater perturbation penetrance, and more consistent editing efficiency. This modular strategy provides a simple approach for conditional CRISPR genome editing that preserves guide integrity and should be readily adaptable to time-resolved functional genomics and pooled screening applications.

cell biology↗

bioRxiv2024

A benchmark comparison of CRISPRn guide-RNA design algorithms and generation of small single and dual-targeting libraries to boost screening efficiency

Genome-wide CRISPR sgRNA libraries have emerged as transformative tools to systematically probe gene function. While these libraries have been iterated over time to be more efficient, their large size limits their use in some applications. Here, we benchmarked publicly available genome-wide single-targeting sgRNA libraries and evaluated dual targeting as a strategy for pooled CRISPR loss-of-function screens. We leveraged this data to design two minimal genome-wide human CRISPR-Cas9 libraries that are 50% smaller than other libraries and that preserve specificity and sensitivity, thus enabling broader deployment at scale.

molecular biology↗

bioRxiv2023

Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

The cytoplasmic dynein-1 (dynein) motor plays a key role in cellular organisation by transporting a wide variety of cellular constituents towards the minus ends of microtubules. However, relatively little is known about how the biosynthesis, assembly and functional diversity of the motor is orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a guide RNA library targeting 18,253 genes, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints generated from multiplexed images revealed co-functional genes involved in many cellular processes, including several candidate novel regulators of core dynein functions. Mechanistic analysis of one of these proteins, the RNA-binding protein SUGP1, provides evidence that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our dataset represents a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organisation that were captured by our high-content imaging.

cell biology↗