RNA detection and transcription-dependent editing with reprogrammed tracrRNAs
AssigneesHelmholtz-Zentrum FüR Infektionsforschung GmbH, Julius-Maximilians-UniversitäT WüRzburg
InventorsChase Beisel, Chunlei Jiao, Cynthia Mira Sharma, Gaurav Dugur
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Abstract
The present invention relates to methods for detecting at least one sensed RNA in a cell, tissue, and/or sample using at least one non-naturally occurring tracrRNA specifically hybridizing with said sensed RNA and at least one tracrRNA-dependent CRISPR nuclease enzyme binding to at least one target nucleic acid, as well as respective systems and diagnostic and therapeutic uses thereof.
Claims19
A method for detecting, in a cell, tissue, and/or sample, at least one sensed RNA that hybridizes to a tracrRNA that is not a crRNA, said method
- comprising:
- a) contacting said cell, tissue and/or sample with at least one non-naturally occurring tracrRNA comprising an anti-repeat portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid;
- wherein said at least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of at least one RNA-cleaving enzyme;
- b) detecting binding of said at least one tracrRNA-dependent CRISPR nuclease enzyme to said at least one target nucleic acid;
- wherein binding detects said at least one sensed RNA in said cell, tissue and/or sample.
A method for recording transcription of at least one target DNA in a cell, tissue and/or sample, said method comprising:
- a) contacting said sample, cell and/or tissue with at least one non-naturally occurring tracrRNA comprising an anti-repeat portion that specifically hybridizes with a first portion of a sensed RNA that is not a crRNA, wherein a second portion of said sensed RNA specifically hybridizes with said at least one target DNA;
- wherein said least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of at least one RNA-cleaving enzyme;
- and
- b) nicking or cleaving of said at least one target nucleic acid in said sample by said nuclease,
- c) detectably editing said at least one target DNA comprising;
- and
- d) optionally, detecting said editing of said at least one target DNA;
- e) wherein detecting said editing of said at least one target DNA records transcription of said at least one sensed RNA in said cell, tissue and/or sample.
A method for detecting, in a cell, tissue, and/or sample, at least one sensed RNA that hybridizes to a tracrRNA that is not a crRNA, said method comprising:
- a) contacting said cell, tissue, and/or sample with at least one non-naturally occurring tracrRNA comprising an anti-repeat portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid;
- wherein said at least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of at least one RNA-cleaving enzyme;
- and
- b) detecting cleavage of said at least one target nucleic acid in said sample by said nuclease enzyme;
- wherein detecting said cleavage of at least one target nucleic acid detects said at least one sensed RNA in said cell, tissue and/or sample.
A method for detecting, in a cell, tissue, and/or sample, at least one sensed RNA that hybridizes to a tracrRNA that is not a crRNA, said method comprising:
- a) contacting said cell, tissue and/or sample with at least one non-naturally occurring tracrRNA comprising an anti-repeat portion that specifically hybridizes with a first portion of said sensed RNA, wherein a second portion of said sensed RNA specifically hybridizes with at least one target nucleic acid;
- wherein said least one non-naturally occurring tracrRNA is hybridized or is able to be hybridized with said sensed RNA in the presence of at least one tracrRNA-dependent CRISPR nuclease enzyme, optionally further comprising the presence of at least one RNA-cleaving enzyme;
- b) nicking or cleaving of said at least one target nucleic acid in said sample by said nuclease,
- c) detectably editing said at least one target nucleic acid, and
- d) detecting said editing of said at least one target nucleic acid;
- wherein detecting said editing of said at least one target nucleic acid detects said at least one sensed RNA in said cell, tissue and/or sample.
Dependencies are inferred from the claim text.