Chemically-inducible dimerization systems are essential tools to interrogate and control biological systems. To date, the number of such systems is still limited, which impedes their broader application. Moreover, currently available chemically-inducible dimerization systems lack the properties desired for use in therapeutic applications as some small molecules can be toxic to human cells, while some proteins originate from other organisms and may trigger an immune response. To address these problems, we engeenered novel chemically-inducible dimerization systems based on split human proteins that selectively bind small molecules. Using this approach, we developed two efficient dimerization systems based on the split Lyn kinase, which binds the inhibitor dasatinib, as well as the single-chain variable fragment of an anti-nicotine antibody. We demonstrated the use of the designed chemically-inducible dimerization systems for dasatinib and nicotine inducible gene activation in mammalian cells in combination with CRISPR/dCas9 system. We believe that the chemically-inducible systems prepared in this MSc thesis will significantly expand the synthetic biology toolbox. Moreover, the engineering principle used in this MSc thesis is universal and could be used to create novel chemically-inducible systems based on almost any small molecule-binding protein.
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