English Summary
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) loci and CRISPR-associated (Cas) proteins system (CRISPR/Cas) has emerged as an attractive targeted genome editing tools within the last five years. This RNA-guide technology has been applied to introduce multiplex genome modifications to a wide range of cells and organisms. In this application, I repurpose the CRISPR/Cas system for targeted gene activation, called CRISPRa. The CRISPRa will be created by fusing a catalytically inactive Cas9 protein (dCas9) to a ubiquitous transcription activation domain VP64. Integration deficient lentiviral vehicles and a dual-luciferase assay will be chosen to deliver CRISPRa to the targeted cells and analyse the effect of CRISPRa-mediated gene activation, respectively. As an example to elaborate the potential applications of the CRISPRa system, I will apply this system to generate induced pluripotent stem cells from human somatic cells.
Purpose and Perspective:
Current methods for generating induced pluripotent stem cells commonly involve either ectopic expression of the exogenous transcription factors, or delivery of corresponding exogenous mRNA or proteins into the targeted cells. The purpose of this project is to modify the CRISPR/Cas9 system for targeted gene activation and generating human iPSCs. I expect that, with the successful generation of the CRISPRa system, this technology will be applied not only for cell engineering (generating iPSCs) but also for many other potentially biomedical applications such as functional genomics (e.g. investigating the effects of certain pathway activation on cellular functions; endogenous regulatory RNA activation; epigenetic regulator activation and so on) and gene therapy (e.g. activation of cancer repressing genes such as BRCA1, TPTEN, APC and so on).
Danish Summary:
Den lavineagtige tekniske udvikling har muliggjort total kortlægning af menneskers individuelle
arvemasse. Vi står således ofte med den ”genetiske instruktionsbog” i hånden, men vi mangler viden
til at fortolke informationen. For at lære at ”læse instruktionsbøgerne” har vi desperat behov for nye
metoder til funktionelt at udforske og afprøve de ekstremt komplekse genetiske systemer.Den
aktuelle ansøgning foreslår udvikling af et nyt system (CRISPRa) for målrettet aktivering af specifikke
gener med henblik på funktionelle studier.CRISPRa-systemet vil også få praktisk betydning. I første
omgang vil vi designe systemet til udvikling og styring af inducerede pluripotente stamceller (iPSC)
som kan bruges til studier af cellefornyelse og differentiering samt organudvikling og reparation.Der
er fantastiske fremtidige muligheder for at bruge denne teknologi til ”celleterapi” og regenerativ
medicin i forebyggelse og behandling af f.eks. kroniske, degenerative folkesygdomme som
sukkersyge, åreforkalkning, neurodegeneration (præsenil demens) og cancer.