Fig. 1: Schematic of Casilio.
a In Casilio, gRNA is appended with one or more binding sites for Pumilio/FBF (PUF) RNA binding domain-tethered effectors. Each PUF domain contains 8 peptide subunits each programmable to recognize one of the four RNA bases. A collection of PUF-effectors can be created by fusing different effectors to PUF programmed to recognize different 8-mer PUF binding sequence (PBS). Effectors are recruited to dCas9/gRNA-PBS complex according to the fused PUF domain. b Casilio architecture allows multiple molecules of the same or different effectors (E1, E2, …, etc.) to be recruited to a target (multimerization or complex formation) as well as different set of effectors to be recruited at different targets in the same cell (multiplexing). This system is expandable to potentially over 65,000 possible effectors. c Casilio-Imaging capitalizes on the multimerization capability to recruit 15 or more fluorescent proteins to a target as opposed to requiring target site tiling of dCas9-GFP direct fusions using 12 or more individual gRNAs in previous studies. d The potential for Casilio to achieve multimodal operations simultaneously, for example, demethylating histone (using LSD1 module), acetylating histone (CBP), methylating DNA (DNMT3A), activating (p65HSF1) different targets, and at the same time monitoring multiple locations with different fluorescent proteins in live cells. dCas9/gRNA-PBS not drawn for simplicity.
