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Crispr effector system based diagnostics

An effector protein, detection system technology, applied in ICT adaptation, genetic engineering, microbial assay/inspection, etc., can solve problems such as limited operator availability, dependence, low sensitivity, etc.

Pending Publication Date: 2019-11-26
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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  • Crispr effector system based diagnostics
  • Crispr effector system based diagnostics
  • Crispr effector system based diagnostics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0547] Example 2 - Highly sensitive and specific detection of C2C2-mediated DNA and RNA from LEPTOTRICHIA WADEI

[0548] Rapid, cheap, and sensitive nucleic acid tests could facilitate pathogen detection, genotyping, and disease surveillance at the point of care. The RNA-guided, RNA-targeting CRISPR effector Cas13a (formerly known as C2c2) exhibits a "collateral effect" of promiscuous RNase activity upon target recognition. Applicants combined the parallel effect of Cas13a with isothermal amplification to create a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. Applicants used this Cas13a-based molecular detection platform, called SHERLOCK (Specific High Sensitivity Enzyme Reporter Unlocked), to detect specific strains of Zika virus and dengue virus, differentiate pathogenic bacteria, genotype human DNA, and identify Cell tumor DNA mutation. In addition, SHERLOCK reaction reagents can ...

Embodiment 3

[0660] Example 3 Characterization of Cas13b Orthologs with Orthogonal Base Preferences

[0661] Applicants biochemically characterized 14 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 83 A and 85). Applicants were able to heterologously express 14 Cas13b orthologs in E. coli and purify the proteins for in vitro RNase activity assays ( Figure 86 ). Because different Cas13 orthologs may have different base preferences for optimal cleavage activity, Applicants generated a fluorescent RNase homopolymer sensor composed of 5 A, G, C, or U to assess orthogonal cleavage preference. Applicants incubated each ortholog with its cognate crRNA targeting the synthetic 173nt ssRNA 1 and measured parallel cleavage activity using a homopolymer fluorescent sensor ( Figure 83 B and 87).

Embodiment 4

[0662] Example 4 Motif Discovery Screening Using Libraries

[0663] To further explore the diversity of cleavage preferences of various Cas13a and Cas13b orthologs, Applicants developed a library-based approach to characterize motifs of endonuclease activity preference in response to parallel activity. Applicants used a degenerate 6-mer RNA reporter flanked by a constant DNA handle, which allows amplification and readout of the uncut sequence ( Figure 83 C). Incubation of the library with parallel activated Cas13 enzymes resulted in detectable cleavage and was dependent on the addition of the target RNA ( Figure 88 ). Sequencing of the depleted motif revealed an increase in the skewness of the library over the digestion time ( Figure 89 A), base preference is indicated, and selection of sequences above a threshold ratio yields enriched sequences corresponding to cleavage by the enzyme ( Figure 89 B). Sequence logos from enriched motifs reproduced the U preference obse...

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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] Statement Regarding Federally Funded Research [0002] This invention was made with government support under grant numbers MH100706 and MH110049 awarded by the National Institutes of Health and grant number HDTRA1-14-1-0006 awarded by the Defense Threat Reduction Agency. The government has certain rights in this invention. technical field [0003] The subject matter disclosed herein generally relates to rapid diagnostics associated with the use of CRISPR effector systems. Background technique [0004] 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 portable platform has the potential to revolutionize the diagnosis and monitoring of many diseases, provide valuable epidemiological information, and serve as a universal scientific tool. Although many methods for detecting nucleic acids have been developed (Du et al., 2017; Green et al., 2014; Kumar et al., 2...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/689C12Q1/6895G01N33/68G01N33/569G01N33/53C12N15/09C12N15/10C12N15/11
CPCC12N9/22C12Q1/6816C12Q1/6811Y02A50/30C12Q2521/301C12Q2525/301C12Q2537/101C12Q2565/1015C12Q2525/143C12Q2525/205C12Q1/68C12Q1/6806C12Q1/6809C12Q1/6869C12Q1/6876C12N15/113C12N2310/16C12N2310/20C12P19/34G01N33/00
Inventor O·阿布达耶J·J·柯林斯J·戈滕贝格F·张E·S·兰德
Owner THE BROAD INST INC
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