Ross-Thriepland, D. | bioRxiv Science

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↗