Abstract

The RNA-guided DNA endonuclease system CRISPR-Cas9 has been exploited for

genome editing in various species. The nuclease-deficient mutant dCas9 protein can,

when coupled with sgRNAs, bind specific genomic loci without inducing DNA cleavage,

thus serving as a programmable DNA binding protein. To extend the utility of the dCas9

system, we have taken advantage of the ability of Pumilio PUF domains to bind specific

8-mer RNA sequences. By combining these two systems, we established the Casilio

system, which allows for specific and independent delivery of effector proteins to

specific genomic loci. We demonstrated that the Casilio system enables independent upand

down-regulation of multiple genes, as well as live-cell imaging of multiple genomic

loci simultaneously. Importantly, multiple copy of PUF binding sites can be incorporated

on sgRNA backbone, therefore allowing for local multimerization of effectors. In

addition, the PUF domain can be engineered to recognize any 8-mer RNA sequence,

therefore enabling the generation and simultaneous operation of many Casilio modules.

A website specifically for Casilio is at http://casil.io