Biology subjects

Wessels, H.-H.

Publications and source records attributed to Wessels, H.-H..

17 recordsLinked to original sources

bioRxiv2024

Paired CRISPR screens to map gene regulation in cis and trans

Recent massively-parallel approaches to decipher gene regulatory circuits have focused on the discovery of either cis-regulatory elements (CREs) or trans-acting factors. Here, we develop a scalable approach that pairs cis- and trans-regulatory CRISPR screens to systematically dissect how the key immune checkpoint PD-L1 is regulated. In human pancreatic ductal adenocarcinoma (PDAC) cells, we tile the PD-L1 locus using [~]25,000 CRISPR perturbations in constitutive and IFN{gamma}-stimulated conditions. We discover 67 enhancer- or repressor-like CREs and show that distal CREs tend to contact the promoter of PD-L1 and related genes. Next, we measure how loss of all [~]2,000 transcription factors (TFs) in the human genome impacts PD-L1 expression and, using this, we link specific TFs to individual CREs and reveal novel PD-L1 regulatory circuits. For one of these regulatory circuits, we confirm the binding of predicted trans-factors (SRF and BPTF) using CUT&RUN and show that loss of either the CRE or TFs potentiates the anti-cancer activity of primary T cells engineered with a chimeric antigen receptor. Finally, we show that expression of these TFs correlates with PD-L1 expression in vivo in primary PDAC tumors and that somatic mutations in TFs can alter response and overall survival in immune checkpoint blockade-treated patients. Taken together, our approach establishes a generalizable toolkit for decoding the regulatory landscape of any gene or locus in the human genome, yielding insights into gene regulation and clinical impact.

genomics↗

bioRxiv2024

Low copy CRISPR-Cas13d mitigates collateral RNA cleavage

While CRISPR-Cas13 systems excel in accurately targeting RNA, the potential for collateral RNA degradation poses a concern for therapeutic applications and limits broader adoption for transcriptome perturbations. We evaluate the extent to which collateral RNA cleavage occurs when RfxCas13d is delivered via plasmid transfection or lentiviral transduction and find that collateral activity only occurs with high levels of RfxCas13d expression. Using transcriptome-scale and combinatorial CRISPR pooled screens in cell lines with low-copy RfxCas13d, we find high on-target knockdown, without extensive collateral activity regardless of the expression level of the target gene. In contrast, transfection of RfxCas13d, which yields higher nuclease expression, results in collateral RNA degradation. Further, our analysis of a high-fidelity Cas13 variant uncovers a marked decrease in on-target efficiency, suggesting that its reduced collateral activity may be due to an overall diminished nuclease capability.

genomics↗

bioRxiv2024

Systematic reconstruction of molecular pathway signatures using scalable single-cell perturbation screens

Recent advancements in functional genomics have provided an unprecedented ability to measure diverse molecular modalities, but learning causal regulatory relationships from observational data remains challenging. Here, we leverage pooled genetic screens and single cell sequencing (i.e. Perturb-seq) to systematically identify the targets of signaling regulators in diverse biological contexts. We demonstrate how Perturb-seq is compatible with recent and commercially available advances in combinatorial indexing and next-generation sequencing, and perform more than 1,500 perturbations split across six cell lines and five biological signaling contexts. We introduce an improved computational framework (Mixscale) to address cellular variation in perturbation efficiency, alongside optimized statistical methods to learn differentially expressed gene lists and conserved molecular signatures. Finally, we demonstrate how our Perturb-seq derived gene lists can be used to precisely infer changes in signaling pathway activation for in-vivo and in-situ samples. Our work enhances our understanding of signaling regulators and their targets, and lays a computational framework towards the data-driven inference of an atlas of perturbation signatures.

genomics↗

bioRxiv2023

Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox

Alternative splicing is an essential mechanism for diversifying proteins, in which mature RNA isoforms produce proteins with potentially distinct functions. Two major challenges in characterizing the cellular function of isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design. To address these gaps, we developed and methodically tested a strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions in the mature RNA. We performed a high-throughput essentiality screen, quantitative RT-PCR assays, and PacBio long read sequencing to affirm our ability to specifically target and robustly knockdown individual RNA isoforms. In parallel, we provide computational tools for experimental design and screen analysis. Considering all possible splice junctions annotated in GENCODE for multi-isoform genes and our gRNA efficacy predictions, we estimate that our junction-centric strategy can uniquely target up to 89% of human RNA isoforms, including 50,066 protein-coding and 11,415 lncRNA isoforms. Importantly, this specificity spans all splicing and transcriptional events, including exon skipping and inclusion, alternative 5 and 3 splice sites, and alternative starts and ends.

genomics↗

bioRxiv2023

CPA-Perturb-seq: Multiplexed single-cell characterization of alternative polyadenylation regulators

Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity that is governed by the cleavage and polyadenylation (CPA) regulatory machinery. To better understand how these proteins govern polyA site choice we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 known CPA regulators with a 3 scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a statistical framework to specifically identify perturbation-dependent changes in intronic and tandem polyadenylation, and discover modules of co-regulated polyA sites exhibiting distinct functional properties. By training a multi-task deep neural network (APARENT-Perturb) on our dataset, we delineate a cis-regulatory code that predicts responsiveness to perturbation and reveals interactions between distinct regulatory complexes. Finally, we leverage our framework to re-analyze published scRNA-seq datasets, identifying new regulators that affect the relative abundance of alternatively polyadenylated transcripts, and characterizing extensive cellular heterogeneity in 3 UTR length amongst antibody-producing cells. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation in vitro and in vivo.

genomics↗

bioRxiv2022

Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq

Pooled CRISPR screens coupled with single-cell RNA-sequencing have enabled systematic interrogation of gene function and regulatory networks. Here, we introduce Cas13 RNA Perturb-seq (CaRPool-seq) which leverages the RNA-targeting CRISPR/Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable array which is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes. We compared CaRPool-seq to existing Cas9-based methods, highlighting its unique strength to efficiently profile combinatorially perturbed cells. Finally, we apply CaRPool-seq to perform multiplexed combinatorial perturbations of myeloid differentiation regulators in an acute myeloid leukemia (AML) model system and identify extensive interactions between different chromatin regulators that can enhance or suppress AML differentiation phenotypes.

genomics↗

bioRxiv2021

Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells

RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to transiently modulate gene expression outcomes. However, protein and CRISPR RNA (crRNA) delivery in human cells can be challenging and knockdown can be transient due to rapid crRNA degradation. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes enables transient gene expression modulation in primary CD4+ and CD8+ T-cells. This system represents a robust and efficient method to transiently modulate transcripts without genetic manipulation.

bioengineering↗

bioRxiv2020

Scalable pooled CRISPR screens with single-cell chromatin accessibility profiling

Pooled CRISPR screens have been used to identify genes responsible for specific phenotypes and diseases, and, more recently, to connect genetic perturbations with multi-dimensional gene expression profiles. Here, we describe a method to link genome-wide chromatin accessibility to genetic perturbations in single cells. This scalable, cost-effective method combines pooled CRISPR perturbations with a single-cell combinatorial indexing assay for transposase-accessible chromatin (CRISPR-sciATAC). Using a human and mouse species-mixing experiment, we show that CRISPR-sciATAC separates single cells with a low doublet rate. Then, in human myelogenous leukemia cells, we apply CRISPR-sciATAC to target 21 chromatin-related genes that are frequently mutated in cancer and 84 subunits and cofactors of chromatin remodeling complexes, generating chromatin accessibility data for ~30,000 single cells. Using this large-scale atlas, we correlate loss of specific chromatin remodelers with changes in accessibility -- globally and at the binding sites of individual transcription factors. For example, we show that loss of the H3K27 methyltransferase EZH2 leads to increased accessibility at heterochromatic regions involved in embryonic development and triggers expression of multiple genes in the HOXA and HOXD clusters. At a subset of regulatory sites, we also analyze dynamic changes in nucleosome spacing upon loss of chromatin remodelers. CRISPR-sciATAC is a high-throughput, low-cost single-cell method that can be applied broadly to study the role of genetic perturbations on chromatin in normal and disease states.

genomics↗

bioRxiv2020

Transcriptome-wide Cas13 guide RNA design for model organisms and viral RNA pathogens

CRISPR-Cas13 mediates robust transcript knockdown in human cells through direct RNA targeting. Compared to DNA-targeting CRISPR enzymes like Cas9, RNA targeting by Cas13 is transcript- and strand-specific: It can distinguish and specifically knock-down processed transcripts, alternatively spliced isoforms and overlapping genes, all of which frequently serve different functions. Previously, we identified optimal design rules for RfxCas13d guide RNAs (gRNAs), and developed a computational model to predict gRNA efficacy for all human protein-coding genes. However, there is a growing interest to target other types of transcripts, such as noncoding RNAs (ncRNAs) or viral RNAs, and to target transcripts in other commonly-used organisms. Here, we predicted relative Cas13-driven knock-down for gRNAs targeting messenger RNAs and ncRNAs in six model organisms (human, mouse, zebrafish, fly, nematode and flowering plants) and four abundant RNA virus families (SARS-CoV-2, HIV-1, H1N1 influenza and MERS). To allow for more flexible gRNA efficacy prediction, we also developed a web-based application to predict optimal gRNAs for any RNA target entered by the user. Given the lack of Cas13 guide design tools, we anticipate this resource will facilitate CRISPR-Cas13 RNA targeting in common model organisms, emerging viral threats to human health, and novel RNA targets.

genomics↗

bioRxiv2020

Characterizing the molecular regulation of inhibitory immune checkpoints with multi-modal single-cell screens

The expression of inhibitory immune checkpoint molecules such as PD-L1 is frequently observed in human cancers and can lead to the suppression of T cell-mediated immune responses. Here we apply ECCITE-seq, a technology which combines pooled CRISPR screens with single-cell mRNA and surface protein measurements, to explore the molecular networks that regulate PD-L1 expression. We also develop a computational framework, mixscape, that substantially improves the signal-to-noise ratio in single-cell perturbation screens by identifying and removing confounding sources of variation. Applying these tools, we identify and validate regulators of PD-L1, and leverage our multi-modal data to identify both transcriptional and post-transcriptional modes of regulation. In particular, we discover that the kelch-like protein KEAP1 and the transcriptional activator NRF2, mediate levels of PD-L1 upregulation after IFN{gamma} stimulation. Our results identify a novel mechanism for the regulation of immune checkpoints and present a powerful analytical framework for the analysis of multi-modal single-cell perturbation screens.

genomics↗

bioRxiv2020

The m6A reader Ythdf restricts axonal growth in Drosophila through target selection modulation of the Fragile X mental retardation protein

N6-methyladenosine (m6A) regulates a variety of physiological processes through modulation of RNA metabolism. The modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6A alters fly behavior albeit the underlying mechanism and the role of m6A during nervous system development have remained elusive. Here we find that impairment of the m6A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6A reader in the nervous system being required for limiting axonal growth. Mechanistically, we show that Ythdf directly interacts with Fragile X mental retardation protein to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6A pathway controls development of the nervous system by modulating Fmr1 target selection.

molecular biology↗

bioRxiv2020

High-throughput screens of PAM-flexible Cas9 variants for gene knock-out and transcriptional modulation

A key limitation of the commonly-used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site, which reduces the number of accessible genomic loci. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG) but their activity has been measured at only a small number of endogenous sites. Here we devised a high-throughput Cas9 pooled competition screen to compare the performance of both PAM-flexible Cas9 variants and wild-type Cas9 at thousands of genomic loci and across 3 modalities (gene knock-out, transcriptional activation and suppression). We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cutting. Of the PAM-flexible variants, we found that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combined xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants.

bioengineering↗

bioRxiv2019

Principles for rational Cas13d guide design

Type VI CRISPR enzymes have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allow for specific and robust target gene knock-down without altering the genome. However, we currently lack information about optimal Cas13 guide RNA designs for high target RNA knock-down efficacy. To close this gap, we conducted four massively-parallel Cas13 screens targeting the mRNA of a destabilized green fluorescent protein (GFP) transgene and CD46, CD55 and CD71 cell surface proteins in human cells. In total, we measured the activity of 24,460 guide RNA including 6,469 perfect match guide RNAs and a diverse set of guide RNA variants and permutations with mismatches relative to the target sequences. We find that guide RNAs show high diversity in knock-down efficiency driven by crRNA-specific features as well as target site context. Moreover, while single mismatches generally reduce knock-down to a modest degree, we identify a critical region spanning spacer nucleotides 15 - 21 that is largely intolerant to target site mismatches. We developed a computational model to identify guide RNAs with high knock-down efficacy. We confirmed the models generalizability across a large number of endogenous target mRNAs and show that Cas13 can be used in forward genetic pooled CRISPR-screens to identify essential genes. Using this model, we provide a resource of optimized Cas13 guide RNAs to target all protein-coding transcripts in the human genome, enabling transcriptome-wide forward genetic screens.

genomics↗

bioRxiv2018

Global identification of functional microRNA::mRNA interactions in Drosophila

MicroRNAs (miRNAs) are key mediators of post-transcriptional gene expression silencing. Although Drosophila has been of critical importance for miRNA discovery, biogenesis and function, there has been no comprehensive experimental annotation of functional miRNA target sites. To close this gap, we generated the first in vivo map of miRNA::mRNA interactions in Drosophila melanogaster, making use of crosslinked nucleotides in Argonaute (AGO) crosslinking and immunoprecipitation (CLIP) experiments that enable an unambiguous assignment of miRNAs to AGO binding sites at much higher signal-to-noise ratio than computational predictions alone.\n\nAbsolute quantification of cellular miRNA levels showed the miRNA pool in Drosophila cell lines to be more diverse than previously reported. Benchmarking two different CLIP approaches, we identified a similar predictive potential to unambiguously assign thousands of miRNA::mRNA pairs from AGO1 interaction data at unprecedented depth. Quantitative RNA-Seq and subcodon-resolution ribosomal footprinting data upon AGO1 depletion enabled the determination of miRNA-mediated effects on target expression and translation. We thus provide the first comprehensive resource of miRNA target sites as well as their quantitative functional impact in Drosophila.

systems biology↗

bioRxiv2018

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a first global RNA-bound proteome of human HEK293 cells and Salmonella Typhimurium as a bacterial species.

molecular biology↗

bioRxiv2018

Deciphering human ribonucleoprotein regulatory networks

RNA-binding proteins (RBPs) control and coordinate each stage in the life cycle of RNAs. Although in vivo binding sites of RBPs can now be determined genome-wide, most studies typically focused on individual RBPs. Here, we examined a large compendium of 114 high-quality transcriptome-wide in vivo RBP-RNA cross-linking interaction datasets generated by the same protocol in the same cell line and representing 64 distinct RBPs. Comparative analysis of categories of target RNA binding preference, sequence preference, and transcript region specificity was performed, and identified potential posttranscriptional regulatory modules, i.e. specific combinations of RBPs that bind to specific sets of RNAs and targeted regions. These regulatory modules encoded functionally related proteins and exhibited distinct differences in RNA metabolism, expression variance, as well as subcellular localization. This integrative investigation of experimental RBP-RNA interaction evidence and RBP regulatory function in a human cell line will be a valuable resource for understanding the complexity of post-transcriptional regulation.

genomics↗

bioRxiv2017

omniCLIP: Bayesian identification of protein-RNA interactions from CLIP-Seq data

High-throughput immunoprecipitation methods to analyze RNA binding protein - RNA in-teractions and modifications have great potential to further the understanding of post-tran-scriptional gene regulation. Due to the differences between individual approaches, each of a diverse number of computational methods can typically be applied to only one specific se-quencing protocol. Here, we present a Bayesian model called omniCLIP that can be applied to data from all protocols to detect regulatory elements in RNAs. omniCLIP greatly sim-plifies the data analysis, increases the reliability of results and paves the way for integrative studies based on data from different sources.

bioinformatics↗