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A CRIPSR-based genome editing tool for advanced gene therapies

The discovery of the CRISPR/Cas system was awarded the 2020 Nobel Prize in Chemistry in 2020. Researchers from the National Institute of Chemistry report on an important advance of this technology, applicable to fields of biotechnology and personalized medicine, such as treatment of some types of cancer and genetic disease. Results of their study have been published in the journal Nature Communications.

The CRISPR/Cas system is a bacterial immune defense mechanism used to fight phage infections, which has been tailored for use as a genome editing tool, allowing precise, site specific modifications of genomic DNA. The CRISPR system has been vastly used in the field of life sciences in general and in the study of disease mechanisms and the development of new therapeutic approaches for treatment of genetic diseases and some forms of cancer. The two-component system is composed of a Cas protein and a small RNA molecule. The Cas endonuclease is guided by the RNA molecule to a specific site in the genome which may be edited.  More than 60 clinical studies employing the CRISPR technology are currently underway for treatment of a plethora of different diseases.

Researchers from the Department of Synthetic Biology and Immunology at the National Institute of Chemistry have attempted to further improve the CRISPR system for gene knock-out through tethering the Cas9 endonuclease via coiled-coil forming peptides to an exonuclease, an enzyme which allows recession of gDNA at the Cas9 target site. They termed the system CCExo. Dimerization of the coiled-coil forming peptides allows co-localization of the exonuclease at the double-stranded break site, leading to larger DNA lesions, which in turn lead to a greater frequency of gene knock-out events.  CCExo was shown to have a robust effect on several different cell lines and genomic targets. Dr. Duško Lainšček, the leading researcher of the study, stated: 'We recognized therapeutic potential of such a system for treatment of genetic diseases, which we have successfully demonstrated on a mouse model of chronic myeloid leukemia, where we have specifically targeted a genetic lesion present only in cancerous, but not healthy cells.' With this approach they managed to specifically eradicate cancerous cells, leaving cells without the specific genetic signature intact. Experiments on cells from cancer patient donors where carried out in collaboration with prof. Matjaž Sever, prof. Helena Podgornik and PhD student Veronika Mikolič from the Department of Hematology at the University Medical Centre Ljubljana.

The research was carried out at the Department of Synthetic Biology and Immunology at the National Institute of Chemistry by leading authors dr. Duško Lainšček and dr. Vida Forstnerič under the supervision of prof. Roman Jerala, in connection to projects funded by the European Research Council (ERC) MaCChines and PoC, led by prof. Roman Jerala and projects funded by the Slovenian Research Agency. Prof. Jerala  believes that the results of this study represent an important technological advance for personalized therapeutic approaches, which are one of the leading research topics of his group at the National Institute of Chemistry and which they wish to further pursue through establishment of a center for gene and cell therapy in collaboration with clinicians, among others prof. Samo Zver, head of the Department of Hematology, in order to bring state of the art therapies to Slovenian patients.

Authors of the research: Duško Lainšček, Vida Forstnerič, Veronika Mikolič, Špela Malenšek, Peter Pečan, Mojca Benčina, Matjaž Sever, Helena Podgornik, Roman Jerala



Authors of the research (from left to right): dr. Roman Jerala, dr. Duško Lainšček, dr. Vida Forstnerič, dr. Mojca Benčina, Peter Pečan, Špela Malenšek, Veronika Mikolič. dr. Matjaž Sever and dr. Helena Podgornik from UKC LJ are missing on the picture.

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