The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy type 1.

Myotonic dystrophy type I (DM1) is a multisystemic autosomal-dominant inherited human disorder that is caused by CTG microsatellite repeat expansions (MREs) in the 3' untranslated region of DMPK. Toxic RNAs expressed from such repetitive sequences can be eliminated using CRISPR-mediated RNA targeting, yet evidence of its in vivo efficacy and durability is lacking. Here, using adult and neonatal mouse models of DM1, we show that intramuscular or systemic injections of adeno-associated virus (AAV) vectors encoding nuclease-dead Cas9 and a single-guide RNA targeting CUG repeats results in the expression of the RNA-targeting Cas9 for up to three months, redistribution of the RNA-splicing protein muscleblind-like splicing regulator 1, elimination of foci of toxic RNA, reversal of splicing biomarkers and amelioration of myotonia.

Peptide Brush Polymers for Efficient Delivery of a Gene Editing Protein to Stem Cells.

The scarcity of effective means to deliver functional proteins to living cells is a central problem in biotechnology and medicine. Herein, we report the efficient delivery of an active DNA-modifying enzyme to human stem cells through high-density cell penetrating peptide brush polymers. Cre recombinase is mixed with a fluorophore-tagged polymer carrier and then applied directly to induced pluripotent stem cells or HEK293T cells.

Self-Transfecting Micellar RNA: Modulating Nanoparticle Cell Interactions via High Density Display of Small Molecule Ligands on Micelle Coronas.

The intracellular delivery of synthetic nucleic acids represents a major challenge in biotechnology and in biomedicine. Methods to deliver short, double-stranded RNA to living cells are of particular interest because of the potential to engage the RNA interference machinery and to regulate mRNA expression. In this work, we describe novel RNA-polymer amphiphiles that assemble into spherical micellar nanoparticles with diameters of ca.

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.

Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs.

Programmable RNA Tracking in Live Cells with CRISPR/Cas9.

RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S.

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

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.

Identification of novel long noncoding RNAs underlying vertebrate cardiovascular development.

Background: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development.

Methods And Results: By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells.

Intracellular mRNA regulation with self-assembled locked nucleic acid polymer nanoparticles.

We present an untemplated, single-component antisense oligonucleotide delivery system capable of regulating mRNA abundance in live human cells. While most approaches to nucleic acid delivery rely on secondary carriers and complex multicomponent charge-neutralizing formulations, we demonstrate efficient delivery using a simple locked nucleic acid (LNA)-polymer conjugate that assembles into spherical micellar nanoparticles displaying a dense shell of nucleic acid at the surface. Cellular uptake of soft LNA nanoparticles occurs rapidly within minutes as evidenced by flow cytometry and fluorescence microscopy.

Alternative splicing in stem cell self-renewal and diferentiation.

This chapter provides a review of recent advances in understanding the importance of alternative pre-messenger RNA splicing in stem cell biology. The majority of transcribed pre-mRNAs undergo RNA splicing where introns are excised and exons are juxtaposed to form mature messenger RNA sequences. This regulated, selective removal of whole or portions of exons by alternative splicing provides avenues for control of RNA abundance and proteome diversity.