Programmable RNA binding proteins for imaging and therapeutics

While RNA was once viewed as a simple intermediate in the Central Dogma, it has now become apparent that RNA provides a critical layer of control to genetic information flow. The lifetime, location, trafficking, and function of RNAs are tightly regulated. Therefore, methods to study the spatial and temporal dynamics of RNAs in physiological conditions are sorely needed. In two recent papers, Abudayyeh et al.¹ and Cox et al.² discovered new Cas family proteins that can be used to deliver protein cargoes to native RNA transcripts in live cells.

The most common method used to obtain spatial information about RNA is fluorescence in situ hybridization (FISH). RNA FISH relies on the annealing of an oligonucleotide probe that uses Watson-Crick-Franklin base pair interactions to selectively deliver a fluorophore to the target transcript. Recently, RNA FISH was improved by leveraging Illumina-based “sequencing by synthesis” technology to yield fluorescent in situ sequencing (FISSEQ). FISSEQ is based on reverse transcription and cDNA amplification of crosslinked nucleic acids in fixed cells, thereby providing location and abundance information of hundreds of RNA transcripts. However, RNA FISH and FISSEQ can only provide a snapshot of RNA location as they generally require cell fixation.

Acquisition of spatial and temporal information of a target RNA in live samples requires the introduction of an exogenous sequence to the RNA of interest. For example, RNA can be fused to the MS2 hairpin, which can then be detected by binding to a GFP-MS2 fusion protein. The development of fluorescent RNA aptamers simplifies these experiments by eliminating the need to express a detector protein fusion. However, these methods require alteration and overexpression of the target RNA, which likely alter the dynamics being measured.

To study native RNA localization in live cells, fluorescently labeled nucleic acids or molecular beacons can be introduced into cells by microinjection. Alternatively, sequence-specific RNA binding proteins, such as Pumillo proteins, can be fused to imaging modalities to track native RNAs. However, the requirement to reprogram the Pumillo binding domains for each individual target transcript limits the practicality of this method. Overall, the field has been limited by a lack of easily programmable RNA binding proteins.

The discovery and characterization of the CRISPR system has revolutionized nucleic acid targeting. Aside from its utility as a programmable nuclease, the CRISPR/Cas9 system has been developed into a reliable and versatile tool to deliver protein cargoes site-selectively to the genome. By fusing cargoes to nuclease-inactivated versions of Cas9, a multitude of epigenetic regulatory and fluorescent proteins have been delivered site-specifically. Up until recently, most work has focused on Cas9 systems that target DNA. Only last year, Nelles et al. reported that the widely-used spCas9 system can be adapted to target RNA in live cells by supplementing a small piece of DNA that forms a duplex with the target RNA to provide the required PAM binding site.³ The resulting RNA-targeting Cas9 (RCas9) system can be used to deliver fluorescent proteins to target RNA and allows imaging of endogenous RNA in live cells. In a recent extension of the RCas9 system, Batra et al. developed a site-specific RNA endonuclease by fusing RCas9 to the PIN RNA nuclease domain.⁴ However, the need for an additional PAMmer DNA presents some potential challenges and limitations of the RCas9 approach. In 2015, Abudayyeh et al. reported the discovery and characterization of the first native RNA-targeting single subunit Cas protein, Cas13a (previously C2c2), and showed that it can be programmed to bind a target RNA.⁵

Now, Abudayyeh et al. report the successful development of a Cas13a-based programmable RNA-guided RNA-targeting platform for use in live mammalian cells.¹ First, the authors performed a screen of Cas13a orthologues and found that the protein variant from Leptotrichia wadei (LwaCas13a) was the most active. The authors showed that endogenous genes in multiple human cell lines and in rice can be knocked down using Cas13a targeting. Knockdown efficiencies were transcript-dependent with maximal knockdown of 85% and 75% for KRAS and PPIB, respectively. Since LwaCas13a can process its own guiding sequence from a CRISPR repeat array, multiple spacer sequences interspersed with repeat regions can be introduced for multiplexed targeting. To assess the selectivity of the LwaCas13a system, the authors performed transcriptome-wide mRNA sequencing, revealing no measurable off-target knockdown.

Abudayyeh et al. further explored whether LwaCas13a could be used as a programmable RNA binding protein for live-cell RNA tracking. The authors developed a catalytically-inactive ‘dead’ LwaCas13a (dLwaCas13a), which maintained robust RNA binding but no longer cleaves the target transcript. To assess whether dLwaCas13a can be used to track RNAs in live cells, Abudayyeh et al. fused the protein to GFP and monitored whether the fusion was exported from the nucleus due to mRNA binding. Nuclear export was observed only when the Cas13a fusion was supplied with a target guide(g)RNA binding to a transcript that gets exported from the nucleus. Moreover, the dLwaCasd13a system could be used to visualize stress granules.

In a follow-up study, Cox et al. identified a related Cas13b variant that is smaller and works analogously to LwaCas13a.² They again showed efficient transcript-specific knockdown and developed an optimized catalytically-inactive Cas13b (dCas13b). To show the versatility of their system, Cox et al. fused the known A-to-I editing enzyme ADAR to dCas13b and reported site-specific deamination. Because inosine is processed analogous to guanosine in cells, this approach allows for an exciting new way to correct disease-relevant mutations at the RNA level.

The development of Cas13a and Cas13b variants as versatile and programmable tools to both cleave target RNA and deliver protein cargoes site-selectively in live cells is an important advance for the field. Like all RNA binding protein-based approaches, there is a possibility that the imaging agent will perturb the native state of the target transcript. In the case of Cas13a, this is potentially a substantial concern, as the protein is quite large (~140 kDa). The protein’s size also presents other obstacles, such as an inability to be delivered efficiently by adenovirus systems. The Cas13b system is smaller (~110 kDa) and can be packaged for viral delivery, but is still a large protein complex. In future work, the in cellulo RNA binding properties of Cas13 proteins need to be carefully assessed in order to understand the capabilities and limitations of each. Overall, the ability to target RNA directly with genetically-encoded tools and without alteration of the target RNA is a milestone for RNA biology. If the previous five years of DNA-targeting Cas9 technologies are predictive, these new RNA-targeting programmable systems will revolutionize our ability to study and perturb RNAs in endogenous environments.