US 12,735,741 B2 · Compositions, methods and uses for quantifying transcription and biosensing of small molecules using a novel type VI CRISPR-Cas assay

Abstract

Embodiments of the instant disclosure relate to novel compositions and methods for analyte detection by quantifying transcription of RNA using CRISPR-based approaches. In certain embodiments, constructs of use in assays disclosed herein can include a novel double-stranded DNA sequence having at least one transcriptional promoter, at least one regulatory element, and a target sequence. In some embodiments, constructs disclosed herein can be used in a system for quantifying transcriptional output in a sample in order to detect presence of an analyte. In other embodiments, constructs and systems disclosed herein can be used to measure enzyme activity, to detect and/or quantify at least one agent that modulates transcription, detect and/or quantify analytes (e.g. a contaminant, biomarkers or other agent) or a combination thereof in accordance with assessing health of a subject or assessing environmental conditions or contaminants.

Claims14

Dependent claims

A system for quantifying transcriptional output in a sample, the system comprising:

at least one type VI CRISPR-Cas effector protein capable of binding a guide RNA;
at least one guide RNA (crRNA);
at least one double-stranded DNA sequence, wherein the at least one double-stranded DNA sequence comprises at least one transcriptional promoter, at least one regulatory element, and at least one target sequence;
at least one fluorescently labeled RNA oligonucleotide comprising a non-target sequence;
and,
at least one RNA polymerase;
wherein the at least one type VI CRISPR-Cas effector protein exhibits collateral RNase activity and cleaves the non-target sequence of the at least one fluorescently labeled RNA oligonucleotide after the at least one type VI CRISPR-Cas effector protein associates with the crRNA and a transcribed target RNA sequence, and
wherein the at least one double-stranded DNA sequence comprises a polynucleotide having at least 85% sequence homology to any one of the polynucleotides represented by SEQ ID NOs:
36-45.

6 more elements

A method for quantifying transcriptional output in a sample comprising:

(a) contacting one or more samples with:
a composition comprising;
at least one type VI CRISPR-Cas effector protein;
at least one guide RNA (crRNA);
at least one double-stranded DNA sequence, wherein the at least one double-stranded DNA sequence comprises a transcriptional promoter, at least one regulatory element, and at least one target sequence;
at least one fluorescently labeled RNA oligonucleotide comprising a non-target sequence;
and,
at least one RNA polymerase;
wherein the at least one type VI CRISPR-Cas effector protein exhibits collateral RNase activity and cleaves the non-target sequence of the at least one fluorescently labeled RNA oligonucleotide after the at least one type VI CRISPR-Cas effector protein associates with the crRNA and a transcribed target RNA sequence, and
wherein the at least one double-stranded DNA sequence comprises a polynucleotide having at least 85% sequence homology to any one of the polynucleotides represented by SEQ ID NOs:
36-45;
and
(b) detecting a signal from cleavage of the non-target sequence, and optionally, measuring at least one of presence, absence and intensity of the signal and quantifying transcriptional output in the one or more samples.

10 more elements

Dependencies are inferred from the claim text.

Compositions, methods and uses for quantifying transcription and biosensing of small molecules using a novel type VI CRISPR-Cas assay

Life of this patent

Compositions, methods and uses for quantifying transcription and biosensing of small molecules using a novel type VI CRISPR-Cas assay

Life of this patent

Abstract

Claims14