Dimēriskas dCas9-FokI sistēmas izveidošana efektīvai zīdītāju šūnu genoma rekombinēšanai
Date
2015
Authors
Berga, Egija
Journal Title
Journal ISSN
Volume Title
Publisher
Latvijas Universitāte
Abstract
CRISPR/Cas9 sistēma nodrošina robustu un plaši pielietojamu genoma rekombinēšanas tehnoloģiju, kas paver iespēju zinātniekiem precīzi manipulēt ar specifickiem genoma elementiem, ko izmantot gēnu funkciju pētījumos bioloģijā un slimību attīstībā.
Darba mērķis bija izveidot un optimizēt dCas9-FokI sistēmu, kas nodrošinātu stabilu un specifisku genoma rekombinēšanu cilvēka šūnās. Mēs piedāvājam alternatīvu tehnoloģiju, kas ir balstīta uz RNS vadītu dCas9-FokI sistēmu un piedāvā ievērojamu genoma rekombinēšanas potenciālu ar samazinātu nespecifiskās šķelšanas (off-targeting) efektu. Iegūtie dati liecina, ka šī izveidotā sistēma var tikt efektīvi izmantota genoma rekombinēšanai zīdītāju šūnās. Iegūtie rezultāti var kalpot kā priekšizpētes dati tālākiem pētījumiem, lai palielinātu dCas9-FokI sistēmas efektivitāti un daudzpusīgo pielietojumu.
Darbs tika izstrādāts Dānijā, HEALTH Aarhus Universitātē, asoc.prof. Jacob Giehm Mikkelsen vadītajā laboratorijā Biomedicīnas departamentā laika posmā no 2014.gada februāra līdz 2014.gada jūnijam.
CRISPR/Cas9 system provides a robust and multiplexable genome editing tool that enables researchers to precisely manipulate specific genomic elements, and facilitate the interpretation of target gene function in biology and diseases. The aim of this project was to optimize and describe dimeric dCas9-FokI fusion system for performing robust and highly specific genome editing in human cells. We propose an alternative methodology based on RNA-guided dCas9-FokI fusion that could offer considerable potential for gene targeting and genome editing applications with potentially reduced off-targeting mediated mutations. Here we report that this system efficiently can be used in mammalian cells to facilitate genome editing. Data obtained in this study can serve as a preliminary data for further research to increase the efficiency and versatility of dCas9-FokI fusion. The experimental work has been performed in the laboratory of associate professor Jacob Giehm Mikkelsen at Department of Biomedicine, HEALTH Aarhus University, Denmark during the period from February 2014 to June 2014.
CRISPR/Cas9 system provides a robust and multiplexable genome editing tool that enables researchers to precisely manipulate specific genomic elements, and facilitate the interpretation of target gene function in biology and diseases. The aim of this project was to optimize and describe dimeric dCas9-FokI fusion system for performing robust and highly specific genome editing in human cells. We propose an alternative methodology based on RNA-guided dCas9-FokI fusion that could offer considerable potential for gene targeting and genome editing applications with potentially reduced off-targeting mediated mutations. Here we report that this system efficiently can be used in mammalian cells to facilitate genome editing. Data obtained in this study can serve as a preliminary data for further research to increase the efficiency and versatility of dCas9-FokI fusion. The experimental work has been performed in the laboratory of associate professor Jacob Giehm Mikkelsen at Department of Biomedicine, HEALTH Aarhus University, Denmark during the period from February 2014 to June 2014.
Description
Keywords
Bioloģija , CRISPR/Cas sistēma , genoma rekombinēšana , dCas9-FokI sistēma , gene editing