Compositions and methods for the expression of CRISPR guide RNAS using the H1 promoter
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.
1. A non-naturally occurring CRISPR system comprising a single vector comprising a bidirectional promoter, wherein the bidirectional promoter comprises:
a) control elements that provide for transcription in one direction of at least one nucleotide sequence encoding a CRISPR system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a nucleic acid; and
b) control elements that provide for transcription in the opposite direction of a nucleotide sequence encoding a RNA-directed nuclease,
wherein the gRNA targets and hybridizes with the target sequence and directs the RNA-directed nuclease to the nucleic acid molecule.
2. The system of claim 1 , wherein the target sequence comprises the nucleotide sequence AN 19 NGG, GN 19 NGG, CN 19 NGG, or TN 19 NGG.
3. The system of claim 1 , wherein the RNA-directed nuclease is a Cas9 protein.
4. The system of claim 1 , wherein the target sequence of the nucleic acid is in a cell.
5. The system of claim 4 , wherein the Cas9 protein is codon optimized for expression in the cell and/or is a Type-II Cas9 protein.
6. The system of claim 4 , wherein the cell is a eukaryotic cell optionally selected from the group consisting of (i) a mammalian cell, (ii) a human cell, and/or (iii) a retinal photoreceptor cell.
7. The system of claim 1 , wherein expression of one or more gene products is decreased.