Publication Date
12-30-2024
Journal
Nature Communications
DOI
10.1038/s41467-024-55134-9
PMID
39737993
PMCID
PMC11685949
PubMedCentral® Posted Date
12-30-2024
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
Humans, Gene Editing, Trans-Activators, RNA, Guide, CRISPR-Cas Systems, Homeodomain Proteins, Amyloid Neuropathies, Familial, HEK293 Cells, RNA, Genome, Human, CRISPR-Cas Systems, Mutation, RNA Interference, Genetic Engineering, Genetic engineering, Nucleic-acid therapeutics, CRISPR-Cas9 genome editing
Abstract
Programmable and modular systems capable of orthogonal genomic and transcriptomic perturbations are crucial for biological research and treating human genetic diseases. Here, we present the minimal versatile genetic perturbation technology (mvGPT), a flexible toolkit designed for simultaneous and orthogonal gene editing, activation, and repression in human cells. The mvGPT combines an engineered compact prime editor (PE), a fusion activator MS2-p65-HSF1 (MPH), and a drive-and-process multiplex array that produces RNAs tailored to different types of genetic perturbation. mvGPT can precisely edit human genome via PE coupled with a prime editing guide RNA and a nicking guide RNA, activate endogenous gene expression using PE with a truncated single guide RNA containing MPH-recruiting MS2 aptamers, and silence endogenous gene expression via RNA interference with a short-hairpin RNA. We showcase the versatility of mvGPT by simultaneously correcting a c.3207C>A mutation in the ATP7B gene linked to Wilson's disease, upregulating the PDX1 gene expression to potentially treat Type I diabetes, and suppressing the TTR gene to manage transthyretin amyloidosis. In addition to plasmid delivery, we successfully utilize various methods to deliver the mvGPT payload, demonstrating its potential for future in vivo applications.
Included in
Biochemical Phenomena, Metabolism, and Nutrition Commons, Community Health and Preventive Medicine Commons, Dietetics and Clinical Nutrition Commons, Genetic Phenomena Commons, Genetic Processes Commons, Medical Genetics Commons, Medical Specialties Commons, Neurosciences Commons, Nutrition Commons
Comments
Programmable and modular systems capable of orthogonal genomic and transcriptomic perturbations are crucial for biological research and treating human genetic diseases. Here, we present the minimal versatile genetic perturbation technology (mvGPT), a flexible toolkit designed for simultaneous and orthogonal gene editing, activation, and repression in human cells. The mvGPT combines an engineered compact prime editor (PE), a fusion activator MS2–p65–HSF1 (MPH), and a drive-and-process multiplex array that produces RNAs tailored to different types of genetic perturbation. mvGPT can precisely edit human genome via PE coupled with a prime editing guide RNA and a nicking guide RNA, activate endogenous gene expression using PE with a truncated single guide RNA containing MPH-recruiting MS2 aptamers, and silence endogenous gene expression via RNA interference with a short-hairpin RNA. We showcase the versatility of mvGPT by simultaneously correcting a c.3207C>A mutation in the ATP7B gene linked to Wilson’s disease, upregulating the PDX1 gene expression to potentially treat Type I diabetes, and suppressing the TTR gene to manage transthyretin amyloidosis. In addition to plasmid delivery, we successfully utilize various methods to deliver the mvGPT payload, demonstrating its potential for future in vivo applications.