Aio-Casilio: a robust CRISPR–Cas9–Pumilio system for chromosome labeling

Abstract

The biological function of chromatin bases on its spatial organization and dynamic activities in different situations. Labeling and tracing of genomic sequences has been a huge challenge in studying the spatial dynamics of chromatin. We reported the development of 'all-in-one Casilio system (Aio-Casilio)', a new system that enables the labeling of endogenous genomic loci. The Aio-Casilio system consists of the dCas9 protein, mClover fused with the Pumilio and FBF proteins RNA-binding domain (PUF domain) and an U6-sgRNA appended with multiple PUF-binding site(s). Here we showed that the Aio-Casilio system is robust tool for imaging of repetitive elements in telomeres and major satellite in N2A cells. Furthermore, we developed a PBTon–Aio-Casilio System, which enables the visualization of repetitive sequences in mES cells. However, this system failed to establish a labeled ES cell line when we attempted to establish a stable labeling cell line for living cell image. This Aio-Casilio imaging tool has potential to significantly improve the capacity to study the conformation of chromosomes in living cells.

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