ABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) has become an integral part of the molecular biology toolkit. CRISPRa genetic screens are an exciting high-throughput means of identifying genes the upregulation of which is sufficient to elicit a given phenotype. Activation machinery is continually under development to achieve greater, more robust, and more consistent activation. In this review, we offer a succinct technological overview of available CRISPRa architectures and a comprehensive summary of pooled CRISPRa screens. Furthermore, we discuss contemporary applications of CRISPRa across broad fields of research, with the aim of presenting a view of exciting emerging applications for CRISPRa screening.
ABSTRACT
Although ACE2 is the primary receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here, we use whole-genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe Coronavirus Disease 2019 (COVID-19) infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , COVID-19/genetics , Antiviral Agents/pharmacology , Angiotensin-Converting Enzyme 2/metabolism , Fibroblasts/metabolism , Protein Binding , Membrane Proteins/genetics , Membrane Proteins/metabolismABSTRACT
Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.
Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Exons/genetics , Extracellular Vesicles/metabolism , Nanoparticles/chemistry , RNA, Guide, Kinetoplastida/metabolism , Base Sequence , Cell Survival , Dimerization , Gene Editing , Genetic Vectors/metabolism , HEK293 Cells , HIV Protease/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Ligands , Luciferases/metabolism , RNA Splicing/genetics , RNA, Catalytic/metabolism , Ribonucleoproteins/metabolism , Tissue Donors , tat Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Duchenne muscular dystrophy (DMD) is a monogenic disorder and a candidate for therapeutic genome editing. There have been several recent reports of genome editing in preclinical models of Duchenne muscular dystrophy1-6, however, the long-term persistence and safety of these genome editing approaches have not been addressed. Here we show that genome editing and dystrophin protein restoration is sustained in the mdx mouse model of Duchenne muscular dystrophy for 1 year after a single intravenous administration of an adeno-associated virus that encodes CRISPR (AAV-CRISPR). We also show that AAV-CRISPR is immunogenic when administered to adult mice7; however, humoral and cellular immune responses can be avoided by treating neonatal mice. Additionally, we describe unintended genome and transcript alterations induced by AAV-CRISPR that should be considered for the development of AAV-CRISPR as a therapeutic approach. This study shows the potential of AAV-CRISPR for permanent genome corrections and highlights aspects of host response and alternative genome editing outcomes that require further study.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Muscular Dystrophy, Duchenne/therapy , Animals , Animals, Newborn , CRISPR-Cas Systems/immunology , Dependovirus , Disease Models, Animal , Dystrophin/genetics , Genetic Therapy/methods , Genetic Vectors , Immunity, Cellular/immunology , Immunity, Humoral/immunology , Mice , Mice, Inbred mdx , Muscular Dystrophy, Duchenne/geneticsABSTRACT
Chronic pain afflicts as much as 50% of the population at any given time but our methods to address pain remain limited, ineffective and addictive. In order to develop new therapies an understanding of the mechanisms of painful sensitization is essential. We discuss here recent progress in the understanding of mechanisms underlying pain, and how these mechanisms are being targeted to produce modern, specific therapies for pain. Finally, we make recommendations for the next generation of targeted, effective, and safe pain therapies.
