Crispr double nickase based amplification compositions, systems, and methods

A technology of nicking enzymes and complexes, applied in biochemical equipment and methods, hydrolytic enzymes, genetic engineering, etc., can solve problems such as application limitations and low sensitivity

Pending Publication Date: 2021-04-09
THE BROAD INST INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these isothermal amplification methods may still require an initial denaturation step and multiple sets of primers
Furthermore, novel approaches combining isothermal nucleic acid amplification with portable platforms (Du et al., 2017; Pardee et al., 2016), provided high detection specificity in point-of-care (POC) settings, but were not applied due to low sensitivity some limitations

Method used

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  • Crispr double nickase based amplification compositions, systems, and methods
  • Crispr double nickase based amplification compositions, systems, and methods
  • Crispr double nickase based amplification compositions, systems, and methods

Examples

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Embodiment 1

[0331] Example 1 - CRISPR-Nickase-Based Amplification (CRISPR-NEAR) and NEAR SHERLOCK Detection

[0332] In this example, nicking enzyme-based amplification was tested using a CRISPR-Cas enzyme called CRISPR-NEAR combined with a CRISPR SHERLOCK detection method. figure 1 A schematic diagram of nickase-based amplification using CRISPR-Cas enzymes is shown.

[0333] CRISPR-NEAR can be performed using DNA or RNA input. CRISPR-NEAR is also compatible with downstream SHERLOCK assays by incorporating T7 promoter sequences in the amplification primers. Figure 9 A schematic diagram of the combination of CRISPR-NEAR with the SHERLOCK assay is shown. One of the main advantages of using CRISPR-NEAR is that it can be amplified much faster than RPA. The method uses very simple buffers that allow easy combination of all steps of the SHERLOCK assay into one reaction. On the other hand, RPA amplification uses very viscous buffers that are difficult to use with other reagents.

[0334] ...

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Abstract

The embodiments disclosed herein utilized RNA targeting effectors to provide robust CRISPR-based nucleic acid amplification methods and systems. Embodiments disclosed herein can amplify both double-stranded and single-stranded nucleic acid targets. Moreover, the embodiments disclosed herein can be combined with various detection platforms, for example, CRISPR-SHERLOCK, to achieve detection and diagnostic with attomolar sensitivity. 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 the benefit of U.S. Provisional Application No. 62 / 767,059, filed November 14, 2018, and U.S. Provisional Application No. 62 / 690,278, filed June 26, 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. MH100706, MH110049, and HL141201 awarded by the National Institutes of Health. The government has certain rights in this invention. technical field [0005] The subject matter disclosed herein relates generally to nucleic acid amplification methods, systems and rapid diagnostics related to the use of CRISPR effector systems. Background technique [0006] Nucleic acid is a universal symbol of biological information. The ability to rapidly detect nucleic acids with high sensitivity and single-base specificity...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844
CPCC12Q1/68C12Q1/6844Y02A50/30C12N9/22C12N15/113C12Q2527/101C12Q2527/125C12N2310/20
Inventor F·张M·凯尔纳J·戈滕贝格O·阿布达耶
Owner THE BROAD INST INC
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