Optogenetic control of GGGGCC repeat-containing RNA phase transition

The GGGGCC (G₄C₂) hexanucleotide repeat expansion in the C9ORF72 gene is a major cause of both hereditary amyotrophic lateral sclerosis and familial frontotemporal dementia. Recent studies have shown that G₄C₂ hexanucleotide repeat-containing RNA transcripts ((G₄C₂)_n RNA) could go through liquid-liquid phase separation to form RNA foci, which may elicit neurodegeneration. However, the direct causality between these abnormal RNA foci and neuronal toxicity remains to be demonstrated. Here we introduce an optogenetic control system that can induce the assembly and phase separation of (G₄C₂)_n RNA foci with blue light illumination in human cells, by fusing a specific (G₄C₂)_n RNA binding protein as the linker domain to Cry2, a protein that oligomerizes in response to blue light. Our results demonstrate that a higher number of G₄C₂ repeats have the potential to be induced into more RNA foci in the cells. Both spontaneous and induced RNA foci display liquid-like properties according to FRAP measurements. Computational simulation shows strong consistency with the experimental results and supports the effect of our system to promote the propensity of (G₄C₂)_n RNA towards phase separation. This system can thus be used to investigate whether (G₄C₂)_n RNA foci would disrupt normal cellular processes and lead to pathological phenotypes relevant to repeat expansion disorders.