Biology subjects

Dorighi, K.

Publications and source records attributed to Dorighi, K..

5 recordsLinked to original sources

bioRxiv2025

A novel, high-density CRISPR activation platform for mapping cancer dependencies and resistance pathways ex vivo and in vivo

CRISPR activation (CRISPRa) enables precise, locus-specific upregulation of gene expression, offering potential for both ex vivo and in vivo applications. However, the lack of scalable, high-coverage tools has limited its use in comprehensive genetic screens, particularly in murine models. Here, we introduce Partita, a next-generation, whole-genome CRISPRa sgRNA platform designed for unparalleled efficiency and depth in gene activation studies. Partita employs a high-density targeting strategy, deploying 10 sgRNAs per transcription start site, structured into five gene family-specific sub-libraries to maximize transcriptional induction. To demonstrate its capabilities, we performed a series of large-scale screens: an in vitro enrichment/depletion screen in iBMDMs, whole-genome CRISPRa screens in a double-hit lymphoma model to uncover genes driving resistance to pro-apoptotic drugs (venetoclax, nutlin-3a, etoposide), and an in vivo whole-genome screen identifying accelerators of Myc-driven lymphomagenesis. Each experiment revealed both expected and novel regulators of cellular phenotypes, with a high validation rate in secondary assays. By enabling robust, high-throughput gain-of-function screening, Partita unlocks new avenues for functional genomics and expands the toolkit for discovering key drivers of biological processes across diverse research fields.

cancer biology↗

bioRxiv2025

Deficiency in POLE exonuclease causes synthetic lethality in highly aneuploid cancer cells

Aneuploidy is a hallmark of cancer and is associated with drug resistance and poor clinical outcomes across diverse cancer types. However, no therapies have been clinically established to target highly aneuploid tumors. By analyzing a real-world dataset comprising nearly half a million tumor samples subjected to comprehensive genomic profiling, we identified a striking mutual exclusivity between POLE exonuclease domain mutations and high aneuploidy burden. This observation was independently validated using data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE). Probabilistic modeling revealed that the elevated quantity and unique spectrum of mutations induced by POLE exonuclease deficiency increase the likelihood of inactivating essential genes on chromosome arms harboring losses, leading to a synthetic lethal phenotype in highly aneuploid cells. Functional experiments demonstrated that POLE exonuclease activity is essential for the viability of highly aneuploid cancer cell lines but is dispensable in diploid cells. These findings suggest that selective inhibition of POLE exonuclease may represent a promising therapeutic strategy for targeting highly aneuploid tumors.

cancer biology↗

bioRxiv2024

Degron-modified Cas12a enhances single-cell CRISPR screening

Single-cell CRISPR (Perturb-seq) screens have primarily relied on Cas9 whereas Cas12a, despite its unique effectiveness for multiplex guide expression, remains underexplored. This may be due to Cas12as guide RNA array (pre-crRNA) self-processing activity and the subsequent challenges associated with pre-crRNA sequence recovery. By developing modified pre-crRNA expression vectors and a degron-based Cas12a system, we overcome the self-processing constraint, allowing for accurate detection of pre-crRNAs at the single-cell level, thus greatly expanding possibilities for future Perturb-seq efforts.

molecular biology↗

bioRxiv2024

Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening

Cas12a is a gene-editing tool that simplifies multiplexed gene targeting through its RNase activity, enabling maturation of individual crRNAs from a pre-crRNA-encoding RNA. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene-editing in cells from enAsCas12aKI mice. To test in vivo activity, we transduced haematopoietic stem cells from E-MycT/+;enAsCas12aKI/+animals with Trp53-targeting pre-crRNAs followed by transplantation into irradiated recipient animals. Tumour development was accelerated and TRP53 protein lost. We generated compact, genome-wide Cas12a knockout libraries targeting each gene with four guide RNAs encoded on two (Menuetto) or one (Scherzo) vector. Introducing these libraries into E-MycT/+;enAsCas12aKI/+lymphoma cells followed by treatment with an MCL-1 inhibitor (S63845) or TRP53-inducer (nutlin-3a) identified known and novel drug resistance genes. Finally, we demonstrate simultaneous gene knockouts (Trp53 or combined Bax/Bak) and activation (Cd19) in primary T cells and mouse dermal fibroblasts from crosses of our enAsCas12a and CRISPR activation models (dCas9a-SAM). Our enAsCas12a mouse model and accompanying libraries enhance genome engineering capabilities and complements current CRISPR technologies.

molecular biology↗

bioRxiv2023

Accelerated drug resistant variant discovery with an enhanced, scalable mutagenic base editor platform

Personalized cancer therapeutics bring directed treatment options to patients based on the genetic signatures of their tumors. Unfortunately, tumor genomes are remarkably adaptable, and acquired resistance to these drugs through genetic means is an all-too-frequent occurrence. Identifying mutations that promote resistance within drug-treated patient populations can be cost, resource, and time intensive. Accordingly, base editing, enabled by Cas9-deaminase domain fusions, has emerged as a promising approach for rapid, large-scale resistance variant screening in situ. We adapted and optimized a conditional activation-induced cytidine deaminase (AID)-dCas9 system, which demonstrated greater heterogeneity of edits with an expanded footprint compared to the most commonly utilized cytosine base editor, BE4. When combined with a custom sgRNA library, we were able to identify both individual and complex, compound variants in EGFR and BRAF that confer resistance to established EGFR inhibitors. This system and the developed analytical pipeline provide a simple, highly-scalable platform for cis or trans drug-modifying variant discovery and for uncovering unique insights into protein structure-function relationships.

molecular biology↗