Defects decisively influence the properties of virtually any material. It is therefore desirable to control the occurrence and properties of defects down to the atomic scale. While many methods have been successfully developed to influence defects in an indirect way (e.g. heat treatments, ion implantation, etc...), the direct interaction and control over individual defects is still in its infancy. This method of direct control promises to greatly deepen our understanding of the properties of single defects and may even lead to the discovery of novel physical phenomena. In this work, we demonstrate ways of directly controlling and testing individual defects in the form of dislocations and grain boundaries. Bilayer graphene, being the thinnest material to host extended dislocations, serves as the perfect model material for dislocation manipulation. Using a precisely controlled micromanipulator it is possible to directly interact with individual line defects in situ in scanning electron microscopy1. Besides showcasing fundamental properties of dislocations such as line tension and interaction with free edges, a novel switching reaction at threading dislocations was observed. For the manipulation further developments were made in the form of a mechanical cleaning approach as well as a setup for diffraction in SEM2. Furthermore, using a layered crystal (VSe2) the sliding behavior of twist grain boundaries is analyzed. By cutting and compressing inclined micropillars made from a single-crystalline specimen, twist grain boundaries can be created. After compression, grain boundary sliding can be tested using micromanipulation combined with spring-table based force measurement. Ultra-low sliding friction and self-retraction is observed for twist grain boundaries. Finally, an experimental pathway towards the analysis of the atomic-scale grain-boundary sliding mechanisms in layered systems will be demonstrated. 1. Schweizer, P., Dolle, C. & Spiecker, E. In situ manipulation and switching of dislocations in bilayer graphene. Sci. Adv. 4, (2018). 2. Schweizer, P., Denninger, P., Dolle, C., Rechberger, S. & Spiecker, E. Low Energy Nano Diffraction (LEND) – Bringing true Diffraction to SEM. Microsc. Microanal. 25, 450–451 (2019).