LADL: Light-activated dynamic looping for endogenous gene expression control

Mammalian genomes are folded into tens of thousands of long-range looping interactions1,2. The cause and effect relationship between looping and genome function is poorly understood, and the extent to which chromatin loops are dynamic on short time scales remains a fundamental unanswered question. Currently available strategies for loop engineering involve synthetic transcription factors tethered to dCas93,4or zinc fingers5,6, which are constitutively expressed5,6or induced on long time scales by the presence of a small molecule3.Here we report a new class of 3D optoepigenetic toolsfor the directed rearrangement of 3D chromatin looping on short time scales using blue light. We create synthetic architectural proteins by fusing the CIBN protein subunit from Arabidopsis thaliana with enzymatically dead Cas9 (dCas9). We target our light-activated dynamic looping system (LADL) to two genomic anchors with CRISPR guide RNAs and engineer their spatial co-localization via light-induced heterodimerization of the cryptochrome 2 (CRY2) protein with dCas9-CIBN. We apply LADL to redirect a stretch enhancer(SE)away from its endogenous Klf4target gene and to the Zfp462promoter. Looping changes occur as early as four hours after light induction. Using single molecule RNA FISH, we observe a LADL-induced increase in the total nascent Zfp462transcripts and the number of Zfp462 alleles expressing simultaneously per cell.Moreover, LADL also increased synchronous Sox2 expression after reinforcement of a known Sox2-SElooping interaction. LADL facilitates loop synchronization across a large population of cells without exogenous chemical cofactors and can enable future efforts to engineer reversible and oscillatory looping on short time scales. Overall design: Total of 20 5C samples from 5 independent replicate experiments on LADL-engineered mES cells and negative controls after blue light illumination or in dark.