DNA-binding proteins physically interact with the DNA and directly affect genomic functions. The eukaryotic genome is compacted into chromatin, limiting the DNA access to nuclear factors. In this Ph.D. thesis, I explored the dynamic mechanisms, that allow the target recognition and access to genomic regions occupied by nucleosomes for the genome editor Cas9, the DNA-sensor protein cGAS, and the transcription factor MYC-MAX. For mammalian genome editing applications, the bacterial Cas9 nuclease must target chromatinized DNA. Previous enzymatic studies showed that Cas9 nuclease activity is reduced by the presence of nucleosomes but there is still no systematic understanding of the mechanisms governing Cas9 targeting and nuclease activity in the chromatin context. To investigate if this loss of activity in chromatinized DNA is due to impaired DNA access or due to reduced residence time, I set out to systematically image chromatin invasion by Cas9 in real-time on the single-molecule level. Single-molecule measurements provide detailed mechanistic insight into intricate molecular processes, and are thus suitable to reveal how Cas9 dynamically engages nucleosomes. However, single-molecule experiments, in particular of complex chromatin samples, are difficult to perform and the reproducible and quantitative determination of parameters can be challenging. I addressed this challenge and developed a new method - XSCAN (multiplexed single-molecule detection of chromatin association) - to parallelize dynamic single-molecule observations, where the interactions of dCas9 to many different types of nucleosomes are observed simultaneously in one single-molecule experiment. I provided each nucleosome type with an identifying DNA sequence, called “barcode”, within its nucleosomal DNA. Parallel experiments were subsequently spatially decoded, via the detection of specific binding of dye labelled DNA probes. Using XSCAN, I then revealed that the time required for stable dCas9 binding is greatly increased for target sequences located further within nucleosome structure, as it is coupled to transient DNA unwrapping events at the nucleosome periphery. Moreover, nucleosomes decrease the association rate constant and suppress non-specific binding in vitro by shortening the residence time of dCas9 for sgRNA containing mismatches by up to 3-fold at more internal sites. This study provided the kinetics of nucleosome inhibition of Cas9 nuclease activity which is critical to the success of genome or epigenome editing applications. In the following chapters of this thesis, I further optimized XSCAN and adopted the single-molecule colocalization microscopy techniques to study two other biological questions. First, I explored on the single-molecule level the inhibitory mechanism of the cGAS DNA sensor's self-activation by Barrier-to-autointegration factor. Secondly, to clarify the cryo-EM structure of transcription factor MYC-MAX bound to a nucleosome, I probed the MYC-MAX nucleosome binding on the single-molecule level. Together, the work presented in this Ph.D. thesis shed light on the complex mechanisms of chromatin effectors – DNA/chromatin interactions. Furthermore, the newly established XSCAN is a robust, fast and semi-automated single-molecule technique, which I expect to be used by the scientific community in order to address new questions in chromatin biology.