Engineering inducible biomolecular assemblies for genome imaging and manipulation in living cells

Genome architecture and organization play critical roles in cell life. However, it remains largely unknown how genomic loci are dynamically coordinated to regulate gene expression and determine cell fate at the single cell level. We have developed an inducible system which allows S imultaneous I maging and M anipulation of genomic loci by B iomolecular A ssemblies (SIMBA) in living cells. In SIMBA, the human heterochromatin protein 1α (HP1α) is fused to mCherry and FRB, which can be induced to form biomolecular assemblies (BAs) with FKBP-scFv, guided to specific genomic loci by a nuclease-defective Cas9 (dCas9) or a transcriptional factor (TF) carrying tandem repeats of SunTag. The induced BAs can not only enhance the imaging signals at target genomic loci using a single sgRNA, either at repetitive or non-repetitive sequences, but also recruit epigenetic modulators such as histone methyltransferase SUV39H1 to locally repress transcription. As such, SIMBA can be applied to simultaneously visualize and manipulate, in principle, any genomic locus with controllable timing in living cells. Imaging non-repetitive loci in living cells remains challenging. Here, the authors engineered an inducible system whereby biomolecular assemblies can be guided to specific genomic loci by a nuclease-defective Cas9, allowing the simultaneous imaging and manipulation of the loci.