NOMIS Awardee Adriano Aguzzi and colleagues have generated two human genome-wide arrayed libraries, which represent a powerful resource for the individual perturbation of human protein-coding genes. Their findings were published in a bioRxiv preprint on May 25, 2022.
Genome-wide CRISPR phenotypic screens are clarifying many fundamental biological phenomena. While pooled screens can be used to study selectable features, arrayed CRISPR libraries extend the screening territory to cell-nonautonomous, biochemical and morphological phenotypes. Using a novel high-fidelity liquid-phase plasmid cloning technology, we generated two human genome-wide arrayed libraries termed T.spiezzo (gene ablation, 19,936 plasmids) and T.gonfio (gene activation and epigenetic silencing, 22,442 plasmids). Each plasmid encodes four non-overlapping single-guide RNAs (sgRNAs), each driven by a unique housekeeping promoter, as well as lentiviral and transposable vector sequences. The sgRNAs were designed to tolerate most DNA polymorphisms identified in 10,000 human genomes, thereby maximizing their versatility. Sequencing confirmed that ~90% of each plasmid population contained ≥3 intact sgRNAs. Deletion, activation and epigenetic silencing experiments showed efficacy of 75-99%, up to 10,000x and 76-92%, respectively; lentiviral titers were ~10^7/ml. As a proof of concept, we investigated the effect of individual activation of each human transcription factor (n=1,634) on the expression of the cellular prion protein PrPC. We identified 24 upregulators and 12 downregulators of PrPC expression. Hence, the T.spiezzo and T.gonfio libraries represent a powerful resource for the individual perturbation of human protein-coding genes.
According to Karl Popper, fundamentally new discoveries cannot be rooted in prior knowledge (Popper, 1992). A powerful strategy to circumvent this limitation is to perform experiments that do not rely on priors. Unbiased genetic screens, whose development Popper did not live to see, fulfill this requirement. In the last decades, RNA interference- or mutagen-mediated screenings have greatly improved our understanding of biology and human health and transformed drug development for diseases (Acevedo-Arozena et al., 2008; Boutros and Ahringer, 2008). CRISPR-mediated techniques (Cong et al., 2013; Jinek et al., 2012) have enormously expanded the toolkits of genetic screening, and now allow for gene ablation (CRISPRko), activation (CRISPRa), interference (CRIS-PRi) and epigenetic silencing (CRISPRoff) (Amabile et al., 2016; Gilbert et al., 2014; Hsu et al., 2014; Jinek et al., 2012; Kampmann, 2020; Knott and Doudna, 2018; Nunez et al., 2021; Wang et al., 2016). CRISPR-based screenings yield both overlapping and distinct hits compared to RNA-interference-based screenings, and CRISPR-mediated gene perturbations are much more specific than RNA interference-based methods (Evers et al., 2016; Gilbert et al., 2014; Morgens et al., 2016; Smith et al., 2017). Thus, genome-wide CRISPR-based gene perturbation libraries are of essential importance to identify and understand the underlying biology of genes involved in various biological processes and diseases.
Continue reading this bioRxiv preprint: Robust and Versatile Arrayed Libraries for Human Genome-Wide CRISPR Activation, Deletion and Silencing
Professor of neuropathology and director of the Institute of Neuropathology
University of Zurich
Understanding the Heterogeneity of the SARS-CoV-2 Immune Response
NOMIS RESEARCH PROJECT