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Multi-effector crispr based diagnostic systems

An effector protein, detection system technology, applied in applications, gene therapy, genetic engineering, etc., can solve problems such as limited operator availability, low sensitivity, and high cost

Pending Publication Date: 2020-07-24
THE BROAD INST INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, qPCR methods are sensitive but expensive and rely on complex instrumentation, limiting availability to 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 not applied due to low sensitivity limited

Method used

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  • Multi-effector crispr based diagnostic systems
  • Multi-effector crispr based diagnostic systems
  • Multi-effector crispr based diagnostic systems

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0481]Example 1: Enhancing CAS13 activity with additional CRISPR-associated proteins

[0482] CRISPR effectors often interact with additional components to modulate activity, and applicants seek to exploit these interactions to increase the sensitivity and speed of SHERLOCK. Type VI-B CRISPR systems usually have interference regulatory proteins Csx27 and Csx28, and the co-expression of Csx28 has been shown to increase the interference activity of Cas13b protein in vivo, which means that they can increase the endonuclease activity of Cas13b in vitro. Since Csx28 was unstable in our hands, applicants purified the Csx28-Sumo fusion protein from three type VI-B systems ( Figure 5 A), and tested whether Csx28 supplementation increases the activity of Cas13a and Cas13b proteins. Applicants have found that the Csx28 protein either reduces the activity of Cas13 or increases target-independent (target-independent) cleavage ( Image 6 A-F).

Embodiment 2

[0483] Example 2: Characterization of Csm6 Cleavage Activity

[0484] Interest was aroused by recent studies demonstrating nucleic acid-based allosteric activation of the CRISPR type III effector nuclease Csm6, and applicants wondered whether the endonucleic acid activity of Cas13 could generate a potent activator of Csm6 containing a 2,3-loop Linear RNA adenine phosphate ( Figure 7 C, 7D). Applicants therefore sought to characterize the RNA end chemistry of Cas13-derived cleavage products by performing a Cas13 in vitro cleavage assay on synthetic ssRNA 2 containing A or U homopolymer loops ( Figure 7 A). Post-cleavage fluorescent labeling of in vitro cleavage reactions confirmed that LwCas13a and PsmCas13b produced cleavage products with 5' hydroxylation and 2'3' cyclic phosphate RNA ends ( Figure 7 B). This result led us to express and purify both known and novel Csm6 orthologs for exploring the use of Csm6 together with Cas13 for positive feedback signal amplificatio...

Embodiment 3

[0485] Example 3: Positive feedback signal amplification using CRISPR-Csm6

[0486] To couple Cas13 activity with Csm6 activation, Applicants designed RNA activators that would yield optimal Csm6 stimulation following Cas13 cleavage. Applicants incubated PsmCas13b with longer poly-A activators whose length is shortened by cleavage to generate short, cyclic phosphate-terminated activators. PsmCas13b digestion of the activator resulted in a modest increase in LsCsm6 activity ( Figure 14 A), possibly due to a series of sub-activators resulting from cleavage.

[0487] Applicants improved on this approach by designing RNA activators that contain two base properties (poly-A stretches with optimal activator length, followed by poly-U stretches that can be cleaved by U-targeting Cas13 enzymes). Applicants found that upon addition of the target, LwaCas13a was able to digest these activators and produce optimal activators against EiCsm6 and LsCsm6, and that this activation required t...

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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 both 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 formatfor 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 / 556,408, filed September 9, 2017; U.S. Provisional Application No. 62 / 610,121, filed December 22, 2017; and U.S. Provisional Application No. .62 / 630,808 in equity. The entire contents of the aforementioned applications are incorporated herein by reference in their entirety. [0003] Statement Regarding Federally Funded Research [0004] This invention was made with government support under grant number MH110049 awarded by the National Institutes of Health. The government has certain rights in this invention. technical field [0005] The subject matter disclosed herein generally relates to rapid diagnostics associated with 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 on a portabl...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/00
CPCC12Q1/6816C12N9/22C12N15/11C12Q1/6804C12Q1/6823C12N2320/10C12N2310/20Y02A50/30C12Q2521/301C12Q2563/155C12Q2521/337C12Q2563/125C12Q2521/119C12Q2525/205B01L3/5085B01L2300/123B01L2300/126C12N15/113C12N2800/80G01N33/5308
Inventor F·张J·戈滕贝格O·阿布达耶
Owner THE BROAD INST INC
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