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Crispr system based droplet diagnostic systems and methods

A droplet, microwell technology for chemical instruments and methods, biochemical equipment and methods, laboratory containers, etc., which can solve problems such as dependence, low sensitivity, and limited operator availability

Pending Publication Date: 2021-10-01
THE BROAD INST INC +3
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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, limiting 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 suffer from low sensitivity However, the application is somewhat limited

Method used

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  • Crispr system based droplet diagnostic systems and methods
  • Crispr system based droplet diagnostic systems and methods
  • Crispr system based droplet diagnostic systems and methods

Examples

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Embodiment 1

[0677] This example describes the development of a combinatorial alignment reaction (CARMEN) for multiplex nucleic acid evaluation and the implementation of CARMEN using Cas13 (CARMEN-Cas13). As shown herein, CARMEN-Cas13 specifically, selectively and simultaneously tests dozens of samples of all human-associated viruses with ≥10 sequenced genomes. In addition, CARMEN-Cas13 utilizes the sensitivity and specificity of Cas13 detection to discriminate all strains of different virus species in parallel and detect groups of single nucleotide variants such as drug resistance mutations. In summary, CARMEN-Cas13 is a highly multiplexed CRISPR-based nucleic acid detection platform that enables epidemiological surveillance on an unprecedented scale.

[0678]CARMEN transforms traditional CRISPR-based nucleic acid detection into multiplexed assays by confining each sample and detection mixture in an emulsified droplet and constructing sample-detection mixture pairs in a microwell array ( ...

Embodiment 3

[0907] Example 3: Region-specific detection panels

[0908] In this project, a diagnostic panel will be developed for the virus species and strains prevalent in Honduras. In parallel, applicants will collaborate with the National Autonomous University of Honduras (UNAH) to deploy existing Cas13-based Zika virus detection and dengue serotyping assays to be tested on patient samples. Hardware will be deployed at UNAH for Cas13-based multiplex diagnostics and collaborators will be trained in the technology. Successful achievement of these goals will generate and validate a CRISPR-based multiplex detection technology for disease surveillance in a country with many endemic viruses. This work will be a critical first step toward a world where every infected person entering a hospital receives a molecular diagnosis, receives improved patient care, and is informed by a rich data set about the prevalence of the virus. Contribute to public health efforts.

[0909] The first goal is t...

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Abstract

RNA targeting proteins are utilized to provide a robust massively multiplexed CRISPR-based diagnostic by detection in droplets with attomolar sensitivity. Detection of both DNA and RNA with comparable levels of sensitivity at nanoliter volumes can differentiate targets from non-targets based on single base pair differences, with applications in multiple scenarios in human health including, for example, viral detection, bacterial strain typing, and sensitive genotyping.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Application No. 62 / 767,070, filed November 14, 2018, U.S. Provisional Application No. 62 / 841,812, filed May 1, 2019, and U.S. Provisional Application No. 62 / 871,056, filed July 5, 2019 rights and interests. The entire content of the application identified above is hereby incorporated by reference in its entirety. [0003] Electronic Sequence Listing Citation [0004] The contents of the Electronic Sequence Listing (BROD_3830WP_ST25.txt; size 217KB, creation date October 7, 2019) are incorporated herein by reference in their entirety. technical field [0005] The subject matter disclosed herein relates generally to droplet diagnostics associated with the use of CRISPR systems. Background technique [0006] The ability to rapidly detect nucleic acids with high sensitivity and single-base specificity against a large number of samples in a short period of time has the potential t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6816C12Q1/6825
CPCC12Q1/6883C12Q2600/106C12Q1/6806C12Q2521/301C12Q2563/179C12Q2565/629C12Q2563/107B01L3/502761B01L2200/0652C12Q1/6804C12Q1/701C12Q2600/156C12Q2600/16G01N21/6428G01N2021/6439G01N15/1023
Inventor C·梅尔沃德C·A·弗雷杰H·梅特斯基P·萨贝蒂G·萨库J·克赫C·阿克曼P·布莱尼D·黄
Owner THE BROAD INST INC
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