Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved ...understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which may impact the effectiveness of transcript depletion of target sequences. However, our understanding of the features and corresponding methods which can predict whether a specific sgRNA will effectively knockdown a transcript is very limited.
Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in HeLa cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner.
Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.
All aspects of mRNA lifetime and function, including its stability, translation into protein, and trafficking through the cell, are tightly regulated through coordinated post-transcriptional ...modifications and interactions with a multitude of RNA effector proteins. Despite the increasing recognition of RNA regulation as a critical layer of mammalian gene expression control and its increasing excitement as a therapeutic target, tools to study and control RNA regulatory mechanisms with temporal precision in their endogenous environment are lacking. Here, we present small molecule-inducible RNA-targeting effectors based on our previously developed CRISPR/Cas-inspired RNA targeting system (CIRTS). The CIRTS biosensor platform is based on guide RNA (gRNA)-dependent RNA binding domains that interact with a target transcript using Watson–Crick–Franklin base pair interactions. Addition of a small molecule recruits an RNA effector to the target transcript, thereby eliciting a local effect on the transcript. In this work, we showcase that these CIRTS biosensors can trigger inducible RNA editing, degradation, or translation on target transcripts in a small molecule-dependent manner. We further go on to show that the CIRTS RNA base editor biosensor can induce RNA base editing in a small molecule-controllable manner in vivo. Collectively this work provides a new set of tools to probe the dynamics of RNA regulatory systems and control gene expression at the RNA level.
Long noncoding RNAs (lncRNAs) represent a multidimensional class of regulatory molecules that are involved in many aspects of brain function. Emerging evidence indicates that lncRNAs are localized to ...the synapse; however, a direct role for their activity in this subcellular compartment in memory formation has yet to be demonstrated. Using lncRNA capture-seq, we identified a specific set of lncRNAs that accumulate in the synaptic compartment within the infralimbic prefrontal cortex of adult male C57/Bl6 mice. Among these was a splice variant related to the stress-associated lncRNA, Gas5. RNA immunoprecipitation followed by mass spectrometry and single-molecule imaging revealed that this Gas5 isoform, in association with the RNA binding proteins G3BP2 and CAPRIN1, regulates the activity-dependent trafficking and clustering of RNA granules. In addition, we found that cell-type-specific, activity-dependent, and synapse-specific knockdown of the Gas5 variant led to impaired fear extinction memory. These findings identify a new mechanism of fear extinction that involves the dynamic interaction between local lncRNA activity and RNA condensates in the synaptic compartment.
Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cells at the RNA level. However, both fundamental studies and ...potential translational applications are impeded by a lack of methods to target specific RNAs with effector proteins. Here, we present CRISPR-Cas-inspired RNA targeting system (CIRTS), a protein engineering strategy for constructing programmable RNA control elements. We show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, translational activators, and base editors to target transcripts. We further demonstrate that CIRTS is not only smaller than naturally occurring CRISPR-Cas programmable RNA binding systems but can also be built entirely from human protein parts. CIRTS provides a platform to probe fundamental RNA regulatory processes, and the human-derived nature of CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.
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•CIRTS is a general strategy for engineering programmable RNA effectors•Nucleases, epitranscriptomic regulators, and editors can be delivered by CIRTS•CIRTS can be constructed entirely from human protein “parts” and delivered by AAV•Orthogonal CIRTS can deliver target multiple effectors to different transcripts
Engineered modular RNA-guided RNA-targeting effectors synthesized entirely from human protein parts provide a set of new tools that might overcome the size and immunogenicity limitations of CRISPR-Cas systems.
Chemical modifications to messenger RNA are increasingly recognized as a critical regulatory layer in the flow of genetic information, but quantitative tools to monitor RNA modifications in a ...whole-transcriptome and site-specific manner are lacking. Here we describe a versatile platform for directed evolution that rapidly selects for reverse transcriptases that install mutations at sites of a given type of RNA modification during reverse transcription, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N
-methyladenosine (m
A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m
A sites. The optimal evolved reverse transcriptase enabled detection of well-characterized m
A sites and revealed hundreds of m
A sites in human mRNA. This work develops and validates the reverse transcriptase evolution platform, and provides new tools, analysis methods and datasets to study m
A biology.
RNA has long been an enticing therapeutic target, but is now garnering increased attention, largely driven by clinical successes of RNA interference–based drugs. While gene knockdown by ...well-established RNA interference– and other oligonucleotide-based strategies continues to advance in the clinic, the repertoire of targetable effectors capable of altering gene expression at the RNA level is also rapidly expanding. In this review, we focus on several recently developed bifunctional molecular technologies that both interact with and act upon a target RNA. These new approaches for programmable RNA knockdown, editing, splicing, translation, and chemical modifications stand to provide impactful new modalities for therapeutic development in the coming decades.
Post-transcriptional gene expression regulation of RNA has emerged as a key factor that controls mammalian protein production. RNA trafficking, translation efficiency, and stability are all ...controlled at the transcript level. For example, in addition to the commonly known processing steps of capping, splicing, and polyadenylation, RNA can be chemically modified. In eukaryotes, N 6-methyladenosine (m6A) is the most prevalent mRNA modification. While the writers, erasers, and readers for m6A are rapidly being uncovered and studied at the whole-cell level, their competitive interplay to regulate methylated RNA transcripts has yet to be elucidated. To address this limitation, we report the development of programmable dPspCas13b-m6A reader proteins to investigate the regulatory effects of specific readers on single transcripts in live cells. We fused the two most well-characterized m6A reader proteins, YTHDF1 and YTHDF2, to a catalytically inactive PspCas13b protein, which can target the reader to a specific RNA of interest using guide RNA (gRNA) complementarity. We then demonstrate that the fused reader proteins each retain their reported functional role on a reporter construct: YTHDF2 induces degradation and YTHDF1 enhances translation. Finally, we show that the system can target endogenous mRNA transcripts within cells, using YTHDF2 as an exemplar, where we found tethering with YTHDF2 leads to decay of the target transcript. The development of dCas13b-based tools to study the regulation of endogenous RNAs will dramatically enhance our understanding of how RNA regulation occurs at the single RNA level. Additionally, our new tools, which permit transcript-specific mediated decay or enhanced protein production, will find utility in synthetic biology applications aimed at controlling genetic information flow at the RNA level.
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