Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy

Abstract

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

Faculty Opinions recommendation of Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

“…A High-Throughput Screen to Identify Genes Required for SARS-CoV-2 Infection To identify key genes required for SARS-CoV-2 infection, we performed a genome-scale loss-of-function screen targeting 19,050 genes in the human genome using the GeCKOv2 CRISPR-Cas9 library (Sanjana et al, 2014). The GeCKOv2 library contains 122,411 CRISPR single-guide RNAs (sgRNAs) (6 guide RNAs per gene) and has previously been used in CRISPR screens for drug resistance, immunotherapy, synthetic lethality, mitochondrial disease, and therapeutic discovery for muscular dystrophy (Erb et al, 2017;Jain et al, 2016;Lek et al, 2020;Patel et al, 2017;Shalem et al, 2014). First, we transduced a human alveolar basal epithelial carcinoma cell line (A549) that constitutively expresses ACE2 (referred to as A549 ACE2 ) with an all-in-one lentiviral vector containing Cas9, guide RNAs from the GeCKOv2 human library, and a puromycin resistance gene.…”

Cellular and animal models for facioscapulohumeral muscular dystrophy

“…Although this mRNA-based system cannot be used to study disease genetics, it recapitulates the sporadic expression pattern of DUX4 quite well and reproduces the asymmetric phenotypes associated with FSHD. Thus, it provides an excellent model to test therapeutic compounds ( Lek et al, 2020 ). Furthermore, this system can be used to investigate developmental aspects of the disease, as it provides a single pulse of DUX4 expression early in development, which can cause muscle disorganization and other phenotypes in adult zebrafish ( Pakula et al, 2019 ).…”

“…Both the mRNA injection and transgenic zebrafish models have been used for therapeutic testing of candidate small molecule compounds ( Lek et al, 2020 ). However, given that both models lack the endogenous regulatory regions and genomic context of DUX4 , these models are not ideal for studies of FSHD genetics and/or epigenetics, and cannot be used to evaluate antisense oligonucleotide-based therapeutics that target the untranslated regions of the DUX4 transcript.…”

“…It is likely that these embryonic and non-coding transcripts in the context of muscle cause progressive death of myofibers, as DUX4 expression is toxic in many models of FSHD, including human and murine cell cultures and several animal models ( Block et al, 2013 ; Bosnakovski et al, 2008b ; Dandapat et al, 2014 ; Jagannathan et al, 2016 ; Jones et al, 2016 ; Kowaljow et al, 2007 ; Mitsuhashi et al, 2013 ; Rickard et al, 2015 ; Wallace et al, 2011 ; Wuebbles et al, 2010 ). Cell death appears to occur via p53 (also known as TP53) and caspase 3/7 activation ( Bosnakovski et al, 2008b ; DeSimone et al, 2019 ; Kowaljow et al, 2007 ; Lek et al, 2020 ; Wallace et al, 2011 ), although p53-independent mechanisms have been proposed ( Bosnakovski et al, 2017b ; Shadle et al, 2017 ). Exactly how DUX4 triggers cell death has been a subject of much investigation, and evidence exists for the involvement of many pathways, including oxidative stress ( Barro et al, 2010 ; Bosnakovski et al, 2008b ; Bou Saada et al, 2016 ; Cheli et al, 2011 ; Dmitriev et al, 2016 ; Sharma et al, 2013 ; Turki et al, 2012 ; Winokur et al, 2003a ), mRNA processing and quality control ( Feng et al, 2015 ; Rickard et al, 2015 ), impairment of the ubiquitin/proteasome pathway ( Homma et al, 2015 ), aggregation of the nuclear proteins TDP-43 and FUS and disruption of nuclear PML bodies and SC35 speckles ( Homma et al, 2015 , 2016 ), accumulation of toxic double-stranded RNAs ( Shadle et al, 2017 ; Shadle et al, 2019 ), hyaluronic acid signaling ( DeSimone et al, 2019 ) and hypoxia/HIF1α pathways ( Lek et al, 2020 ).…”

“…Cell death appears to occur via p53 (also known as TP53) and caspase 3/7 activation ( Bosnakovski et al, 2008b ; DeSimone et al, 2019 ; Kowaljow et al, 2007 ; Lek et al, 2020 ; Wallace et al, 2011 ), although p53-independent mechanisms have been proposed ( Bosnakovski et al, 2017b ; Shadle et al, 2017 ). Exactly how DUX4 triggers cell death has been a subject of much investigation, and evidence exists for the involvement of many pathways, including oxidative stress ( Barro et al, 2010 ; Bosnakovski et al, 2008b ; Bou Saada et al, 2016 ; Cheli et al, 2011 ; Dmitriev et al, 2016 ; Sharma et al, 2013 ; Turki et al, 2012 ; Winokur et al, 2003a ), mRNA processing and quality control ( Feng et al, 2015 ; Rickard et al, 2015 ), impairment of the ubiquitin/proteasome pathway ( Homma et al, 2015 ), aggregation of the nuclear proteins TDP-43 and FUS and disruption of nuclear PML bodies and SC35 speckles ( Homma et al, 2015 , 2016 ), accumulation of toxic double-stranded RNAs ( Shadle et al, 2017 ; Shadle et al, 2019 ), hyaluronic acid signaling ( DeSimone et al, 2019 ) and hypoxia/HIF1α pathways ( Lek et al, 2020 ). DUX4 is also associated with a number of other cellular phenotypes that may contribute to pathology, such as myoblast differentiation/fusion defects and altered morphology ( Banerji et al, 2018 ; Barro et al, 2010 ; Bosnakovski et al, 2008b , 2017c , 2018 ; Dandapat et al, 2014 ; Knopp et al, 2016 ; Tassin et al, 2012 ; Vanderplanck et al, 2011 ; Winokur et al, 2003b ; Yip and Picketts, 2003 ), altered β-catenin signaling ( Banerji et al, 2015 ), changes to proteomes ( Celegato et al, 2006 ; Jagannathan et al, 2019 ; Tassin et al, 2012 ) and an altered myogenic program ( Bosnakovski et al, 2008b , 2017c ;…”