CRISPR - CAS 13 MEDIATED RNA-BASED DIAGNOSTIC FOR INFECTIOUS DISEASES

Abstract

The present invention relates to a CRISPR-Cas 13-based RNA diagnostic system designed for the rapid and sensitive detection of infectious diseases. This system leverages the RNA-targeting capability of Casl3 enzymes to identify specific viral or bacterial RNA sequences in patient samples. The diagnostic approach involves guide RNAs (gRNAs) tailored to bind to pathogenspecific RNA regions, triggering Casl3-mediated collateral cleavage of reporter molecules, which produces a detectable signal. The method allows for point-of-care testing with minimal equipment, enabling rapid diagnosis in field settings or resource-limited areas. The system is designed for high sensitivity, detecting low levels of viral or bacterial RNA, and can be adapted to various infectious agents, including emerging pathogens. This novel diagnostic platform can significantly improve early detection, reduce transmission, and provide rapid response during outbreaks of infectious diseases.

Specification

1. Permeable to the description: :
The following specification particularly describes the invention and 'manner in which' it'is to be performed.
2. Description:
FIELD OF INVENTION
CRISPR-Cas arrangements are a progressive tool in microscopic physical science, originally found as one the adjusting immune whole in microorganisms. Among lhese systems, CRISPR-Casl3 is specifically important cause it targets RNA a suggestion of correction DNA. This characteristic create it an ideal candidate for expanding condition proposed at detecting RNA viruses, such as those being the reason for differing infectious afflictions. The CRISPR-Cas 13 n T fi F F I F F H F Xi hi. ix T ft A Z 1 i z 7 7 4 i 1 s s 4
'method'exists of a guide RNA (gRNA) that addresses" the Cas 13"enzy me to completing RNA sequences. Upon binding to magnetism purpose, Casl3 activates appeal RNase activity, leading ' to the degradation of the aim RNA. This feature maybe controlled for diagnostic purposes by plotting gRNAs specific to pathogenic RNA sequences guide catching ailments.
BACKGROUND OF THE INVENTION
.Monkey pox virus (MPXV) is an infectious DNA virus with zoonotic characteristics, and socioconneli is a cassette orthopox virus. More virulent strains of Central Africa are associated with greater morbidity and mortality over the West African. In the people's population, monkey pox can also be passed on through contact with infected person 's or animal's respiratory droplets, fluid and skin lesions. Hence regarding the MPXV there is a need to develop fast, inexpensive and yet reliable diagnostics. Though qPCR and other techniques are resource intensive and hence requires expensive instruments and skilled technicians which makes this methods to be limited in many developing countries. This has spawned the innovation of other novel methods of diagnosis like this CRISPR based diagnostics. This RNA-based diagnostics of monkeypox employing CRISPR-Cas 13 is rapid, sensitive, specific, easy-to-use, doesn't break the bank, and works with all types of samples tested in point of care settings which are suited for the management of monkey pox outbreaks.
OBJECTIVE OF THE INNOVATION:
The design of CRISPR-Cas 13 system for CRISPR-Cas 13 mediated RNA based diagnostics targets the need of creating novel rapid, sensitive and accurate molecular based detection technologies to identify RNA virus related diseases that pose a clinical threat.This is because the guide RNAs offer precise targeting for the RNA and therefore they can work on detection of even trace amounts of signals with high specificity.
The system was designed such that it can switch to other pathogens detection with no technical changes other than the guide RNA in the assay.The objective of inventing CRISPR-Casl3 mediated RNA based diagnostics is such that rapid, sensitive, specific, versatile, cost effective, available, as well as scalable options for RNA virus detection are made possible.
SUMMARY OF THE INVENTION:
CRISPR-Casl3 has potential uses in therapeutics in addition to diagnostics. As a kind of antiviral therapy, researchers are looking into how it might be used to specifically target and destroy viral RNAs within infected cells. CRISPR-Cas 13 may play a vital role in future research on infectious £ ^isease-diagnosfic^and h'eatmeiit^approaclies^This novefrnetHoci offers'a subsfantial improvement in the quick diagnosis of infectious diseases, giving medical practitioners the information they need in a responsible way to manage patients and contain outbreaks.
DISCLOSURE OF THE INVENTION:
A member of the class 2 CRISPR systems, CRISPR-Cas 13 targets and cleaves RNA molecules specifically. Unlike other CRISPR enzymes that target DNA, Cast3 is unique in its ability to bind to RNA sequences complementary to its guide RNA (gRNA) and perform two types of cleavage: cis-cleavage, where it cuts the target RNA, and trans-cleavage, where it non-specifically cleaves other nearby RNA molecules. Two higher eukaryotic and prokaryotic nucleotide-binding (HEPN) domains in the enzyme aid in this activity. Particularly noteworthy are the compact size and high biochemical activity of Casl3d, a subtype of Casl3, which make it appropriate for uses like RNA knockdown in mammalian cells and animal models. The secondary structure of the target RNA affects the specificity of the enzyme, and The Guide RNA (gRNA)
The essential element that guides the Casl3 enzyme to its precise target RNA sequence is the guide RNA (gRNA), also referred to as CRISPR-RNA (crRNA). A complementary spacer sequence to the target RNA sequence is present in the gRNA. Because of this complementarity, the gRNA can attach to the target site with specificity, directing Cas13 to start the cleavage. Avoiding mismatches in crucial regions that could impact binding affinity or nuclease activity is one factor to take into account when designing gRNAs for diagnostic applications. Mismatches in proximal regions, on the other hand, may inhibit nuclease function without affecting binding, whereas those in distal regions can lower binding efficiency.
The Target RNA Sequences:
Certain regions of the genomes of bacteria or viruses are known as target RNA sequences, and CRISPR-Cas 13 systems are used to detect or modify these sequences. To prevent off-target consequences, these sequences need to be carefully chosen based on how unique they are to the pathogen of interest. These sequences are selected from conserved areas of the virus's genome, which are less likely to mutate quiqkly but still permit differentiation between variants through minute sequence differences, for use in diagnostics, such as the detection of SARS-CoV-2 variants.
The Detection Reagents:
Typically, fluorescent reporters or other signal-generating molecules that react when cleaved by activated Casl3 enzymes are used as detection reagents in CRISPR-Cas 13 mediated diagnostics.
. XJfjon .sugces^ful. ^indjng^cm^^ct^yation-(by the target-specific g^bJA, (2ajjl3 performs trans-cleavage on these reporter molecules, resulting in a detectable signal change-often fluorescence-that indicates the presence of the target pathogen's genetic material. These ■ chemicals are essential for translating molecular interactions into quantifiable results that can be used as diagnostic indicators.
3. Claims:
1. A CRISPR-Casl3-based RNA detection system, comprising:
o A guide RNA (gRNA) sequence specifically designed to bind to a target RNA sequence of a pathogen associated with an infectious disease;
o A Casl3 enzyme capable of recognizing and binding the gRNA, initiating collateral cleavage of RNA when bound to the target sequence;
o A reporter molecule that produces a detectable signal upon collateral cleavage by the Casl3 enzyme.
2. The system of claim 1. wherein the pathogen is selected from the group consisting of viruses, bacteria, fungi, or parasites.
3. The system of claim 1. wherein the target RNA sequence is from a viral genome, including but not limited to influenza, coronaviruses, or dengue viruses.
4. A method for detecting infectious diseases using the system of claim 1, comprising:
o Obtaining a biological sample suspected of containing RNA from an infectious • agent;
o Adding the sample to a reaction mixture containing the gRNA, Casl3 enzyme, and reporter molecules;
o Allowing the reaction to occur under conditions promoting gRNA binding to the target RNA, Casl3 activation, and collateral cleavage of the reporter;
o Detecting the signal generated by the cleaved reporter molecule as an indicator of the presence of the target pathogen RNA.
5. A CRISPR-Casl3-based diagnostic kit, comprising:
• A set of gRNAs for detecting RNA from one or more infectious agents;
• A Casl3 enzyme or lyophilized Casl3 reaction mix;
• Reporter molecules configured for fluorescence or colorimetric detection;
• A buffer system optimized for Casl3 function;
h N T * for-u^epryd^egtyig ipjeptipijs disease^ frqjji biologic samples.

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