Applications of Cas9 as an RNA-programmed RNA-binding protein

Abstract

The Streptococcus pyogenes CRISPR-Cas system has gained widespread application as a genome editing and gene regulation tool as simultaneous cellular delivery of the Cas9 protein and guide RNAs enables recognition of specific DNA sequences. The recent discovery that Cas9 can also bind and cleave RNA in an RNA-programmable manner indicates the potential utility of this system as a universal nucleic acid-recognition technology. RNA-targeted Cas9 (RCas9) could allow identification and manipulation of RNA substrates in live cells, empowering the study of cellular gene expression, and could ultimately spawn patient- and disease-specific diagnostic and therapeutic tools. Here we describe the development of RCas9 and compare it to previous methods for RNA targeting, including engineered RNA-binding proteins and other types of CRISPR-Cas systems. We discuss potential uses ranging from live imaging of transcriptional dynamics to patient-specific therapies and applications in synthetic biology.

Keywords: CRISPR-Cas; Cas9; RCas9; RNA biology; RNA targeting; RNA-binding proteins.

Figure 1

S. pyogenes Cas9 and sgRNA complexes bound to DNA or RNA. A: The Cas9:sgRNA complex requires a DNA NGG motif referred to as the protospacer adjacent motif (PAM). In the case of DNA binding, the PAM is supplied by the DNA target itself. The mechanism of DNA targeting by Cas9 is described extensively elsewhere. B: RNA-targeted Cas9 (RCas9) relies upon a short oligonucleotide called the PAMmer to supply the PAM motif. By utilizing a mismatched PAMmer, specificity of RCas9 for RNA while avoiding the encoding DNA is achieved. The PAMmer also carries a 5' overhang which is required to maintain target specificity conferred by the sgRNA. As a result, it is hypothesized that the 5’ end of the PAMmer is at least partially dehybridized from the target RNA as Cas9-mediated unwinding of the PAMmer:target RNA duplex may confer an energetic cost that is recovered when the sgRNA hybridizes the target RNA.

Figure 2

Summary of potential RCas9 application areas. A - D describe means by which RNA fate can be manipulated by the RCas9 system. A: With a nuclease-active version of Cas9, siRNA-intractable RNA targets could be cleaved. B: Conversely, gene expression could be amplified by tethering factors that prevent degradation of target RNAs. C: By fusing Cas9 to a trafficking agent, RNAs could be forced to different sites of action in the cell for local translation or other activities. D: The processing of pre-mRNAs could be modulated by fusing Cas9 with a splicing factor to force differential exon choice. E: Along with altering RNA fate, RCas9 could be used to track RNA abundance in time with split luminescent or fluorescent proteins whose complementation is guided by binding of adjacent Cas9 proteins on RNA. F: Split fluorescent proteins could also be used to reveal rare cells by their RNA content for isolation by FACS and subsequent study. G: Finally, split toxic proteins or proteins that transform prodrugs to their active form could also be complemented in an RNA-dependent manner via fusion to Cas9.