Crispr effector system based multiplex diagnostics

A detection system and motif technology, applied in genetic engineering, recombinant DNA technology, microbial assay/inspection, etc., can solve problems such as expensive, low-sensitivity applications, limited availability, etc.

Pending Publication Date: 2020-10-27
THE BROAD INST INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, qPCR methods are sensitive but expensive and rely on complex instrumentation, which limits the availability of trained operators in laboratory settings
Other approaches, such as novel approaches combining isothermal nucleic acid amplification with portable platforms (Du et al., 2017; Pardee et al., 2016), offer high detection specificity in point-of-care (POC) settings, but are somewhat less sensitive due to low sensitivity. restricted apps

Method used

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  • Crispr effector system based multiplex diagnostics
  • Crispr effector system based multiplex diagnostics
  • Crispr effector system based multiplex diagnostics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1- 1

[0691] Example 1 - General Scheme

[0692] Two ways are provided to perform the C2c2 diagnostic test for DNA and RNA. This approach can also be used with protein detection variants following delivery of detection aptamers. The first is a two-step reaction in which amplification and C2c2 detection are done independently. The second is where everything is combined in one reaction and this is called a two-step reaction. It is important to keep in mind that amplification may not be necessary for higher concentration samples, so it is good to have a separate C2c2 protocol that does not have amplification built in.

[0693] Table 11. CRISPR effectors only - no amplification:

[0694] components Volume (μL) Protein (final 44nM) 2 crRNA (final 12nM) 1 Background target (100ng total) 1 target RNA (variable) 1 RNA sensor probe (125nM) 4 MgCl 2 (final 6mM)

[0695] The reaction buffer is: 40mM Tris-HCl, 60mM NaCl, pH 7.3

[0696] This ...

Embodiment 2

[0710] Example 2 - Highly Sensitive and Specific Detection of C2C2-Mediated DNA and RNA from Ciliophora videides

[0711]Rapid, cheap, and sensitive nucleic acid tests can aid point-of-care pathogen detection, genotyping, and disease surveillance. The RNA-guided RNA-targeting CRISPR effector Cas13a (previously known as C2c2) exhibits "side effects" of promiscuous RNase activity following target recognition. Applicants combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and specificity for single-base mismatches. The applicant used this Cas13a-based molecular detection platform, called SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking), to detect specific strains of Zika and Dengue viruses, and to distinguish pathogens bacteria, genotyping human DNA, and identifying mutations in cell-free tumor DNA. In addition, SHERLOCK reagents...

Embodiment 3

[0828] Example 3 - Characterization of Cas13b Orthologs with Orthogonal Base Preferences

[0829] Applicants biochemically characterized fourteen orthologs of the recently defined Type VI CRISPR-Cas13b family of RNA-guided RNA-targeting enzymes to find new candidates for improved SHERLOCK assay technology ( Figure 83A and Figure 85 ). Applicants were able to heterologously express fourteen Cas13b orthologs in E. coli and purify the proteins for in vitro RNase activity assays ( FIG. 86 ). Because different Cas13 orthologs may have different base preferences for optimal cleavage activity, Applicants generated a fluorescent RNase homopolymer sensor consisting of 5 A, G, C, or U to estimate orthogonal cleavage preference. Applicants incubated each ortholog with its cognate crRNA targeting the synthetic 173nt ssRNA 1 and measured incidental cleavage activity using a homopolymer fluorescent sensor ( Figure 83B and Figure 87).

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Abstract

The embodiments disclosed herein utilized RNA targeting effectors to provide a robust CRISPR-based diagnostic with attomolar sensitivity. Embodiments disclosed herein can detect broth DNA and RNA withcomparable levels of sensitivity and can differentiate targets from non-targets based on single base pair differences. Moreover, the embodiments disclosed herein can be prepared in freeze-dried format for convenient distribution and point-of-care (POC) applications. Such embodiments are useful in multiple scenarios in human health including, for example, viral detection, bacterial strain typing,sensitive genotyping, and detection of disease-associated cell free DNA.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 62 / 610,066, filed December 22, 2017; U.S. Provisional Application No. 62 / 623,546, filed January 29, 2018; U.S. Provisional Application No. 62 / 630,814, filed February 14, 2018 and the benefit of U.S. Provisional Application No. 62 / 741,501, filed October 4, 2018. The entire content of the application identified above is hereby incorporated by reference in its entirety. [0003] Statement Regarding Federally Funded Research [0004] This invention was made with government support under Grant Nos. MH110049 and HL141201 awarded by the National Institutes of Health. The government has certain rights in this invention. [0005] Electronic Sequence Listing Citation [0006] The contents of the Electronic Sequence Listing ("BROD-2445WP.ST25.txt"; 1.8 megabytes in size and date of creation November 27, 2018) are hereby incorporated by reference in their entirety. technic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/6804G01N33/68G01N33/53
CPCC12Q1/6804C12Q1/6823C12N9/22Y02A50/30C12Q2521/301C12Q2525/205C12N2310/20C12N15/11C12N15/115C12N2310/16C12N2800/80C12Q1/6876
Inventor F·张B·蔡彻J·戈滕贝格O·阿布达耶
Owner THE BROAD INST INC
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