Small nuclear RNAs enhance protein-free RNA-programmable base conversion on mammalian coding transcripts.

Endogenous U small nuclear RNAs (U snRNAs) form RNA-protein complexes responsible for eukaryotic processing of pre-mRNA into mature mRNA. Previous studies have demonstrated the utility of guide-programmable U snRNAs in targeted exon inclusion and exclusion. We investigated whether snRNAs can also enhance conversion of RNA bases over state-of-the-art RNA targeting technologies in human cells. When compared to adenosine deaminase acting on RNA (ADAR)-recruiting circular RNAs, we find that guided A>I snRNAs consistently increase adenosine-to-inosine editing efficiency for genes with higher exon counts, perturb substantially fewer genes in the transcriptome, and localize more persistently to the nucleus where ADAR is expressed. A>I snRNAs can also edit pre-mRNA 3' splice sites to promote splicing changes. Finally, snRNA fusions to H/ACA box snoRNAs (U>Ψ snRNAs) increase targeted RNA pseudouridylation efficiency. Altogether, our results advance the protein-free RNA base conversion toolbox and enhance minimally invasive RNA targeting technologies to treat genetic diseases.

Programmable macromolecule-based RNA-targeting therapies to treat human neurological disorders.

Disruptions in RNA processing play critical roles in the pathogenesis of neurological diseases. In this Perspective, we discuss recent progress in the development of RNA-targeting therapeutic modalities. We focus on progress, limitations, and opportunities in a new generation of therapies engineered from RNA binding proteins and other endogenous RNA regulatory macromolecules to treat human neurological disorders.

Engineered U1 snRNAs to modulate alternatively spliced exons.

Alternative splicing accounts for a considerable portion of transcriptomic diversity, as most protein-coding genes are spliced into multiple mRNA isoforms. However, errors in splicing patterns can give rise to mis-splicing with pathological consequences, such as the congenital diseases familial dysautonomia, Duchenne muscular dystrophy, and spinal muscular atrophy. Small nuclear RNA (snRNA) components of the U snRNP family have been proposed as a therapeutic modality for the treatment of mis-splicing. U1 snRNAs offer great promise, with prior studies demonstrating in vivo efficacy, suggesting additional preclinical development is merited. Improvements in enabling technologies, including screening methodologies, gene delivery vectors, and relevant considerations from gene editing approaches justify further advancement of U1 snRNA as a therapeutic and research tool. With the goal of providing a user-friendly protocol, we compile and demonstrate a methodological toolkit for sequence-specific targeted perturbation of alternatively spliced pre-mRNA with engineered U1 snRNAs. We observe robust modulation of endogenous pre-mRNA transcripts targeted in two contrasting splicing contexts, SMN2 exon 7 and FAS exon 6, exhibiting the utility and applicability of engineered U1 snRNA to both inclusion and exclusion of targeted exons. We anticipate that these demonstrations will contribute to the usability of U1 snRNA in investigating splicing modulation in eukaryotic cells, increasing accessibility to the broader research community.

Crosstalk between CRISPR-Cas9 and the human transcriptome.

CRISPR-Cas9 expression independent of its cognate synthetic guide RNA (gRNA) causes widespread genomic DNA damage in human cells. To investigate whether Cas9 can interact with endogenous human RNA transcripts independent of its guide, we perform eCLIP (enhanced CLIP) of Cas9 in human cells and find that Cas9 reproducibly interacts with hundreds of endogenous human RNA transcripts. This association can be partially explained by a model built on gRNA secondary structure and sequence. Critically, transcriptome-wide Cas9 binding sites do not appear to correlate with published genome-wide Cas9 DNA binding or cut-site loci under gRNA co-expression. However, even under gRNA co-expression low-affinity Cas9-human RNA interactions (which we term CRISPR crosstalk) do correlate with published elevated transcriptome-wide RNA editing. Our findings do not support the hypothesis that human RNAs can broadly guide Cas9 to bind and cleave human genomic DNA, but they illustrate a cellular and RNA impact likely inherent to CRISPR-Cas systems.

RNA-targeting CRISPR systems from metagenomic discovery to transcriptomic engineering.

Deployment of RNA-guided DNA endonuclease CRISPR-Cas technology has led to radical advances in biology. As the functional diversity of CRISPR-Cas and parallel systems is further explored, RNA manipulation is emerging as a powerful mode of CRISPR-based engineering. In this Perspective, we chart progress in the RNA-targeting CRISPR-Cas (RCas) field and illustrate how continuing evolution in scientific discovery translates into applications for RNA biology and insights into mysteries, obstacles, and alternative technologies that lie ahead.

Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28.

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.