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Casanova, an Italian writer of the eighteenth century, is still quoted today for his many stories of love with women of the time. The molecular precision tool of the same name, developed by scientists from Heidelberg and Berlin, has something in common with its name at first. He looks for a partner and builds close relationships with her, but frees him as easily. However, the partner here is not a woman, but the programmable gene CRISPR / Cas9, which allows to specifically modify the genome in human cells. The researchers have now published their findings in the journal Nature Methods.
CASANOVA literally means "CRISPR / Cas activation through a new optogenetic process based on anti-CRISPR proteins". The anti-CRISPR proteins are small virus proteins that infect bacteria that can bind to CRISPR scissors. Once linked, the gene scissors are blind and can no longer reach their target sequence in the genome. As a result, the viral genome is protected from Genscher shear attacks.
The researchers around Dr. Dominik Niopek, group leader for synthetic biology at the Institute of Pharmacy and Molecular Biotechnology and the Bioquant Center of the University of Heidelberg, and Prof. Dr. med. Roland Eils, Director of the Digital Health Center of the Berlin Institute of Health Research (BIH) and the Charité University Medical Center and Head of the Health Sciences Unit at the Hospital University of Heidelberg, used anti-CRISPR proteins to rearrange them from the outside using genetic engineering techniques – and can be turned off – with light. To do this, the researchers integrated a molecular light sensor from oats into an anti-CRISPR protein. Subsequently, the researchers used the thus generated hybrid – called CASANOVA – with the scissors of the CRISPR gene in human cell cultures.
"In the dark, CASANOVA effectively binds to the scissors of the CRISPR gene and puts them out of action," explains Niopek. "However, if the blue light strikes the pair of proteins in the cell, the romance ends abruptly.The scissors gene is released from the anti-CRISPR protein and thus becomes active."
Thanks to their method, the researchers of Niopek and Eils were able to deliberately modify the genome sequence in human cells by external illumination. CASANOVA also allowed to activate and deactivate genes by simply pressing a button. Even the scissors binding dynamics of the CRISPR gene to their target sequence in the live cell genome could be followed live by scientists under a microscope.
"CASANOVA is not just an innovative tool for fundamental research, for example, to study the interaction between gene activity and cell behavior.The method could become relevant in the future for particularly precise therapies of genetic diseases, "said Eils.
"The versatility and ease of use of CASANOVA is a key advantage over previous CRISPR / Cas9 control methods," adds Bubeck. With Mareike Hoffmann, a doctoral student at the German Cancer Research Center, he has conducted many crucial experiments in the Niopek and Eils laboratories. Bubeck is a student in the Master's program in Molecular Biotechnology at the University of Heidelberg and co-first author of the publication.
Original publication:
Bubeck *, Hoffmann * et al. (2018): Anti-CRISPR proteins designed for optogenetic control of CRISPR / Cas9. Nature methods. DOI: 10.1038 / s41592-018-0178-9
idw 2018/10
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