Characterization and application of type VI-B RNA-targeting CRISPR systems

Abstract

The ability to modify nucleic acids is critical for establishing the role of genetic and transcribed elements in mediating biological phenotypes. Manipulating endogenous DNA sequences in eukaryotic genomes has been greatly aided by the advent of genome editing technologies that utilize programmable nucleases. DNA nucleases derived from class 2 CRISPR systems, which provide adaptive immunity in prokaryotes through cleavage of nucleic acids using a single, multi-domain, RNA-guided endonuclease, have been particularly useful in this regard because they enable targeting of new sites through simple Watson-Crick base pairing rules. Recent computational studies have uncovered the existence of predicted RNA-targeting class 2 CRISPR systems, suggesting that the power of genome editing techniques might be extended to the level of transcripts. In this thesis, I present work describing the discovery and characterization of a new RNA-targeting class 2 CRISPR system: type VI-B. Using a combination of biochemistry and bacterial genetics, we demonstrated that the predicted nuclease of the VI-B system, Casl3b, is an RNA-guided RNase, whose activity can be modulated by the csx genes that often appear in genetic proximity to casl3b. Next, we characterized the behavior of Casl3b and the related enzymes Casl3a and Casl3c in mammalian cells, identifying orthologs of Casl3a and Casl3b with specific RNA interference activity in mammalian cells. Finally, we showed that catalytically inactive versions of a Casl3b ortholog can direct adenosine-to-inosine deaminase activity to transcripts in human cells when fused to the catalytic domain of ADAR2. Using structure-guided mutagenesis, we created a high-specificity version of this system that can be utilized in research or potentially therapeutic contexts. The description of a Casl3b ortholog that can be used to knockdown or recruit RNA-modifying domains to transcripts in mammalian cells suggests the utility of this technology to interrogate and modify transcript function in diverse contexts.