| Abstract [eng] |
Methods for genomic DNA single loci fluorescent labeling and their dynamics tracking over time are often limited to a few loci. Existing methods such as FROS, ANCHOR allow to obtain high contrast of fluorescent signal. However, their application is limited due to integration of long sequences into the genome. Meanwhile, the CRISPR-Cas system, which is increasingly being applied for chromatin labeling studies, effectively allows labeling only repetitive sequences in the genome, since single loci fluorescent labeling is not efficient due to low contrast of the fluorescent signal. Finally, all existing methods encounters challenges due to rapid photobleaching of fluorescent proteins, which complicates long-term imaging experiments. The aim of this work was to apply the Cas12a protein of Francisella novicida for the fluorescence imaging of Schizosaccharomyces pombe genomic DNA for the first time. Moreover we aimed to start the development of a CRISPR-Cas-based PAINT microscopy imaging method. PAINT microscopy is based on the weak interaction of the Cas ribonucleoprotein (RNP) complex with the target DNA sequence. The weak interaction and rapid dissociation would allow one Cas RNP complex which is fused to a fluorescent protein to be quickly replaced by another complex. Successful implementation of this strategy would be beneficial for long-term chromatin imaging. In this work, a strategy for labeling S. pombe chromatin was developed using the dFnCas12a-GFP11×7 and GFP1-10 two-component fluorescent system. During the work, dFnCas12a-GFP11×7 RNP complex was targeted to repetitive and single loci in the S. pombe genome. Meanwhile, the principle of the PAINT strategy was begun to be tested in a simpler model organism, Escherichia coli, by adapting the Streptococcus pyogenes dCas9 and dFnCas12a proteins. Although the work succeeded in constructing functional CRISPR-Cas systems for fluorescent labeling of E. coli plasmid DNA, it was not possible to optimize the labeling strategy for single and repetitive loci in S. pombe cells using the dFnCas12a-GFP11×7 RNP complex. |
