Crispr effector system based diagnostics

A polypeptide detection system comprising:

• a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind to a trigger RNA;
• a RNA-based masking construct;
• one or more detection aptamers comprising a masked RNA polymerase promoter binding site or a masked primer binding site;
• and
• optionally, nucleic acid amplification reagents.

A method for detecting target nucleic acids in a sample or set of samples, comprising:

• distributing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising a CRISPR system comprising an effector protein, one or more guide RNAs designed to bind to corresponding target molecules, wherein said target molecules comprise somatic mutations, germline mutations, and single nucleic acid polymorphisms (SNP) in mammalian cells, and a RNA-based masking construct;
• incubating the sample or set of samples under conditions sufficient to allow binding of the one or more guide RNAs to one or more target molecules;
• activating the CRISPR effector protein via binding of the one or more guide RNAs to the one or more target molecules, wherein activating the CRISPR effector protein results in modification of the RNA-based masking construct such that a detectable positive signal is generated;
• and
• detecting the detectable positive signal, wherein detection of the detectable positive signal indicates a presence of one or more target molecules in the sample, preferably wherein the sample is a biological sample, more preferably wherein biological sample is a tissue sample, a blood sample, a sera sample, a plasma sample, a saliva sample, a urine sample, a stool sample, a cerebrospinal sample, a sputum sample, lymph, synovial fluid, spinal fluid, or mucous.

A method for detecting polypeptides in a sample or set of samples, comprising:

• distributing a sample or set of samples into a set of individual discrete volumes, the individual discrete volumes comprising peptide detection aptamers, a CRISPR system comprising an effector protein and one or more guide RNAs, a RNA-based masking construct, and polypeptide detection aptamers comprising a masked RNA polymerase site or primer binding site;
• incubating the sample or set of samples under conditions sufficient to allow binding of the peptide detection aptamers to the one or more target molecules, wherein binding of the aptamer to a corresponding target molecule exposes the RNA polymerase binding site or primer binding site resulting in generation of a trigger RNA;
• activating the RNA effector protein via binding of the one or more guide RNAs to the trigger RNA, wherein activating the RNA effector protein results in modification of the RNA-based masking construct such that a detectable positive signal is produced;
• and
• detecting the detectable positive signal, wherein detection of the detectable positive signal indicates a presence of one or more target molecules in a sample, preferably wherein the sample is a biological sample, more preferably wherein biological sample is a tissue sample, a blood sample, a sera sample, a plasma sample, a saliva sample, a urine sample, a stool sample, a cerebrospinal sample, a sputum sample, lymph, synovial fluid, spinal fluid, or mucous.

A method for genotyping an SNP, comprising

• (a) amplifying a nucleic acid sample comprising an SNP to obtain amplified DNAs;
• (b) transcribing the amplified DNAs to obtain target RNAs;
• (c) incubating the target RNAs with a RNA-based masking construct, and a C2c2 effector in complex with a first guide RNA targeting a first variant of SNP, wherein binding of the first guide RNA to the target RNA activates the C2c2 effector to cleave the RNA-based masking construct to produce a first detectable signal, and
• optionally, incubating the target RNAs with a RNA-based masking construct, and a C2c2 effector in complex with a second guide RNA targeting a second variant of SNP, wherein binding of the second guide RNA to the target RNA activates the C2c2 effector to cleave the RNA-based masking construct to produce a second detectable signal;
• and
• (d) detecting the first and optionally the second detectable signals to genotype the SNP in the nucleic acid sample.

A method for detecting a cancer-associated mutation, comprising

• (a) amplifying a cell-free DNA sample to obtain amplified DNAs;
• (b) transcribing the amplified DNAs to obtain target RNAs;
• (c) incubating the target RNAs with a RNA-based masking construct, and a C2c2 effector in complex with a first guide RNA targeting a cancer-associated mutation at a genomic locus, wherein binding of the first guide RNA to the target RNA activates the C2c2 effector to cleave the RNA-based masking construct to produce a first detectable signal, and
• optionally, incubating the target RNAs with a RNA-based masking construct, and a C2c2 effector in complex with a second guide RNA targeting a wild-type sequence at the genomic locus, wherein binding of the second guide RNA to the target RNA activates the C2c2 effector to cleave the RNA-based masking construct to produce a second detectable signal;
• and
• (d) detecting the first and optionally the second detectable signals to detect the cancer-associated mutation in the cell-free DNA sample.