I led the first demonstration that programmable CRISPR/Cas9 can recognize endogenous RNA (RCas9) in live cells (Nelles et al., Cell 2016). By fusing a human endonuclease effector module to RCas9, we can track and eliminate microsatellite repeat expansion RNAs that cause myotonic dystrophy (DM), ALS caused by repeat expansion in C9orf72 (C9ALS), and Huntington’s disease (Batra et al., Cell 2017).

To demonstrate in vivo utility of RCas9 as a therapeutic modality, we delivered adeno-associated viral vector (AAV)-packaged RCas9 in DM1 mouse models. Excitingly, we observed sustained RCas9 expression and reversal of both molecular and myotonia features (Batra et al., Nature Biomedical Engineering 2020), achieving the first in vivo proof-of-concept for RNA-targeting CRISPR/Cas as a form of gene therapy. Our work laid the foundation for RCas approaches to reverse disease-linked post-transcriptional phenomena.

Site-directed RNA editing approaches offer great potential to correct mutations in somatic cells while avoiding permanent off-target genomic edits. We had previously showed that nuclease-dead RNA-targeting CRISPR-Cas systems can recruit functional effector domains to RNA in a programmable fashion. My graduate student Ryan Marina and postdoc Kris Brannan demonstrated, rather surprising, that the Cas9 sgRNA spacer sequences are dispensable for directed RNA editing, indicating that Cas9 can act as a RNA-aptamer binding protein. We also performed the first systematic evaluation of engineered CRISPR/Cas-mediated systems for site-specific RNA editing. We show that all systems are comparable in on-target efficiency and off-target specificity with even the Cas13 RNA editing versions (Marina et al., Cell Reports 2020). Importantly, our study suggests that ADAR fusions or recruitment may have therapeutic limitations.

To simultaneously identify new effector domains of RBPs and assign molecular functions to novel RBPs, we have constructed a library of >800 human RBPs fused to bacteriophage MS2 coat protein (MCP). We recently reported the first and most comprehensive tethering screen to date and discovered ~50 effectors that controlled RNA stability and translation (Luo et al., Nature Structural & Molecular Biology 2020). We also showed that fusion of a novel effector to RCas9 conveys programmable enhancement of mRNA translation. Going forward, we will first discern the effect of each RBP on a specific step of RNA processing by tethering the RBP-MCP fusion to reporters with MS2 RNA-stem loops at various locations within pre- or mature mRNA. Then, domains of candidate proteins that score positive for each reporter are separately tethered to decouple their functional effect from the RNA recognition domains of the RBPs. Ongoing work in my lab will continue to utilize our tethering library and new assays to identify novel RBPs and effector domains that will modulate RNA splicing, stability, translation and localization.