An optogenetic CRISPR-Cas9 system for inducing reversible, three-dimensional chromatin loops to regulate target gene expression.

Mammalian genomes are folded through tens of thousands of chromatin interactions. However, these interactions and the topology of the genome are still poorly understood, especially with respect to the temporality of genome interactions. A primary cause of the limited understanding of genome topology is a lack of tools to study the relationship of genome looping and function.

Dr. Cremins and colleagues at the University of Pennsylvania have designed a new class of 3D optogenetic tools for the directed, reversible rearrangement of 3D chromatin looping on short time scales using blue light. Enzymatically dead Cas9 and the CIBN protein from Arabidopsis thaliana heterodimerized with constitutively expressed CRY2 protein to bridge two genomic loci of interest by application of blue light. This process induces loop formation for the control of genome functions, including, but not limited to, gene expression, replication timing, and genome instability.

Light-activated dynamic looping (LADL) facilitates reversible and rapid loop synchronization for regulating gene expression with spatial and temporal control, without the need for an external chemical dimerizer. This method provides a gateway to understanding the role for 3D genome folding in regulating genome function in healthy human development and during the onset and progression of human disease.

• Reversible activation on rapid time scales
• High signal/noise in assays through synchronization of chromatin topology across large populations of cells in response to light stimulus
• Scalable for in vivo applications
• No exogenous chemical dimerizers

Research tool to enable light-activated nucleic acid topology engineering