Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript.

Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci encoded in a single transcript. To establish multiple functions for multiple loci targets, we fused four RNA hairpins, MS2, PP7, com and boxB, to stem-loops of gRNA (guide RNA) scaffolds, separately. The RNA-hairpin-binding domains MCP, PCP, Com and λN22 were fused with different functional effectors. These paired combinations of cognate-RNA hairpins and RNA-binding proteins generated the simultaneous, independent regulation of multiple target genes. To ensure that all proteins and RNAs are expressed in one transcript, multiple gRNAs were constructed in a tandemly arrayed tRNA (transfer RNA)-gRNA architecture, and the triplex sequence was cloned between the protein-coding sequences and the tRNA-gRNA array. By leveraging this system, we illustrate the transcriptional activation, transcriptional repression, DNA methylation and DNA demethylation of endogenous targets using up to 16 individual CRISPR gRNAs delivered on a single transcript. This system provides a powerful platform to investigate synthetic biology questions and engineer complex-phenotype medical applications.