The cohesin complex (SMC1-SMC3-RAD21) shapes chromosomes by DNA loop extrusion, but individual extrusion trajectories were so far unappreciable in vivo. Here, we site-specifically induced dozens of extrusion trajectories anchored at artificial loading sites in living cells. Extruding cohesin transports loading proteins MAU2-NIPBL over megabase DNA distances to blocking CTCF sites that then loop back to the loading sequences, showing that CTCF-CTCF interactions are unnecessary for stabilized contacts between loop extrusion obstacles. When stalled, cohesin can block other extruding cohesin from either direction. Without RAD21, MAU2-NIPBL exclusively accumulate at loading sites, here genome-wide defined as enhancers. SMC1 now also selectively accumulates here, suggesting that cohesin may load modularly on chromatin. Genes inside high cohesin extrusion trajectories are collectively hindered in transcription. This work characterizes the impact, entry and exit sites of individual cohesin loop extrusion trajectories.
