The invention relates to methods of improving efficacy and enhancing silencing of target genes using Ago2 protein-dsRNA complex (RNP) of the invention.

Described herein are methods for treating a retinal degeneration in a subject, such as Leber's congenital amaurosis (LCA), retinitis pigmentosa (RP), and glaucoma. Also provided herein are methods of altering expression of one or more gene products in a cell, such as a retinal ganglion cell. Such methods may comprise utilizing a modified nuclease system, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising a bidirectional HI promoter and gRNAs directed to retinal degeneration related genes, packaged in a single, compact adeno-associated virus (AAV) particle.

Aptamers that specifically bind to ligands of folinic acid, a folate, and derivatives thereof (which may be referred to herein as ligands) are provided, and compositions and methods of use thereof. The aptamers and switches of the invention provide biological sensing capability for detecting the ligands, and are effective in sensing in vitro and in vivo. By specific sensing of the ligand, the aptamers of the invention provide a means of engineering an inducible gene regulatory system that enables dose-dependent control over gene expression in response to the ligand, in vivo and in vitro.

The presently disclosed subject matter provides compositions and methods for the expression of CRISPR guide RNAs using the H1 promoter. In particular, compositions and methods are provided for the use of the H1 promoter to express CRISPR guide RNA (gRNA) with altered specificity of the 5 nucleotide, as well as use of the H1 promoter sequence as a bidirectional promoter to express Cas9 nuclease and the gRNA simultaneously. Compositions and methods are also provided for the expression and regulation of gRNA expression in vivo through the use of RNA ribozymes and regulatable aptazymes.

The present disclosure is directed to polycistronic guide RNAs, DNA encoding polycistronic gRNA, multiplex CRISPR vectors, a plurality of component DNA fragments for assembly into a DNA encoding a polycistronic gRNA array, a plurality of primer pairs for making a plurality of component DNA fragments to be assembled into a DNA encoding a polycistronic gRNA, and methods of making multiplex CRISPR vectors. The current disclosure is directed to multiplexed CRISPR technologies that have great potential for pathway engineering and genome editing. In the current disclosure describes efficient assembly of tRNA/Csy4/Ribozyme-based gRNA arrays which can be produced in a quick and effective process.

A ligand-responsive hammerhead ribozyme is provided. In some embodiments, the ribozyme comprises: i. a first loop that has been replaced by an RNA aptamer that binds to a ligand; and ii. a second loop comprising a modified sequence, wherein the aptamer and the second loop interact in a ligand-dependent manner and autocatalytic cleavage of the ribozyme is ligand-responsive.

In various embodiments, the invention teaches methods for detecting ribonucleic acid (RNA) molecules that contain certain chemical modifications using sequencing technologies. These modified RNAs are otherwise not readily detected using the commonly used cloning protocols required for sequencing. The method further includes bioinformatics analyses to identify specific RNA species that are modified at high resolution.

Provided are constructs and methods for RNA promoter identification.

Provided are minimal hammerhead ribozymes and catalytic strands thereof. Aspects of the present disclosure include a catalytic strand of a minimal hammerhead ribozyme, the catalytic strand including a catalytic core region, a stem I-forming region, a stem II region, and a stem III-forming region. The catalytic strand hybridizes to a target strand via the stem I-forming region and the stem III-forming region. A nucleotide (e.g., an adenine, cytosine, or a natural or non-natural nucleotide base having hydrogen bond donor and acceptor functionalities at positions analogous to those of adenine or cytosine) present in a stem II loop base pairs with a nucleotide (e.g., uracil or cytosine) at position 1.7 of the target strand. Also provided are compositions that include the catalytic strands, and methods of using the catalytic strands, e.g., in a variety of different applications, as well as kits that find use in practicing embodiments of the methods.

The presently disclosed subject matter provides compositions and methods for the expression of CRISPR guide RNAs using the H1 promoter. In particular, compositions and methods are provided for the use of the H1 promoter to express CRISPR guide RNA (gRNA) with altered specificity of the 5 nucleotide, as well as use of the H1 promoter sequence as a bidirectional promoter to express Cas9 nuclease and the gRNA simultaneously. Compositions and methods are also provided for the expression and regulation of gRNA expression in vivo through the use of RNA ribozymes and regulatable aptazymes.