COMPOSITIONS AND METHODS COMPRISING IMPROVEMENTS OF CRISPR GUIDE RNAS USING THE H1 PROMOTER
First Claim
1. A non-naturally occurring nuclease system comprising one or more vectors comprising:
- a) a promoter operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a DNA molecule or RNA molecule in a cell, and wherein the DNA molecule or RNA encodes one or more gene products expressed in the cell; and
b) a regulatory element operable in a cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease,wherein components (a) and (b) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves the DNA molecule or RNA to alter expression of the one or more gene products.
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Abstract
The presently disclosed subject matter provides compositions and methods comprising improvements of a CRISPR system (e.g. CRISPR associated (Cas) 9 (CRISPR-Cas9, non-Cas9 CRISPR systems). Such compositions may comprise modifications to the H1 promoter region, addition of 5′UTR modifications, different orthologous sequences of the H1 promoter, novel compact bidirectional promoter sequences with both pol II and pol III activity, addition of Kozak consensus sequences, termination sequences, addition of conditional pol II/pol III bidirectional promoter expression, addition of a donor template sequence for correcting mutations, or combinations thereof. Other aspects of the invention relate to modifications to Cas9 through post-transcriptional cell-cycle regulation fusions, engineered partial target sites such that the nuclease can bind without DNA cleavage, auto-regulation sites, and N-terminal modifications to modulate half-life.
1 Citation
153 Claims
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1. A non-naturally occurring nuclease system comprising one or more vectors comprising:
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a) a promoter operably linked to at least one nucleotide sequence encoding a nuclease system guide RNA (gRNA), wherein the gRNA hybridizes with a target sequence of a DNA molecule or RNA molecule in a cell, and wherein the DNA molecule or RNA encodes one or more gene products expressed in the cell; and b) a regulatory element operable in a cell operably linked to a nucleotide sequence encoding a genome-targeted nuclease, wherein components (a) and (b) are located on the same or different vectors of the system, wherein the gRNA targets and hybridizes with the target sequence and the nuclease cleaves the DNA molecule or RNA to alter expression of the one or more gene products. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152)
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153. The method of claim 154, wherein the mammal is human.
Specification