HiCRISPR: molecular typing and detection of resistance genes in target microbes (#2017038)

Background

• The Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas system is a prokaryotic adaptive immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity.
• As a consequence of CRISPR/Cas activity, the Direct Repeat (DR) region of the CRISPR acquires foreign pieces of DNA (spacers) that confer extreme diversity to this region and therefore a molecular signature to the strain that can be used for identification and typing.
• CRISPR/Cas has been found in about 48% of bacteria and 90% of archaea genomes sequenced so far.
• Many pathogenic as well as industrial bacterial species contain CRISPR/Cas systems.
• The CRISPR/Cas typing serves as an important and emerging technique for bacterial typing.
• We developed a novel method for CRISPR/Cas typing as well as for parallel identification of resistance and virulence genes.

The Need

Rapid and highly accurate method for pathogens' subtyping and resistance genes' identification is needed in: public health, human medicine, veterinary medicine, agriculture, research, industry and other settings. Our SPACERome kit ability to generate high throughput CRISPR/cas SPACER profile (SPACERome) can be used for identification, characterization and typing of various complex sample like feces.

Examples:

•  Infection control in the hospital setting,
•  Epidemiological investigation in public health settings,
•  Quality control of industrial bacterial cultures,
•  Emerging bacterial pathogen tracking.
•  Patient follow up after FMT procedure by total spacer profiling (SPACERome).

The Technology and Innovation

1. We developed a complete solution for fast and high throughput bacterial CRISPR typing, antimicrobial resistance & virulence determination.
2. Our novel platform allows for designing of ready to use kits for pathogen, organ and other complex sample.
3. Each primer is connected to one of our novel 11 bp specific proMIDs barcodes.
4. By simultaneous PCR amplification we can amplify and barcode all the spacers and other wanted genes in a single step.
5. The PCR products are later combined to a single DNA library and sequenced by the Illumina MiSeq (or other NGS platform).
6. We developed a Python based program (Spopy) for automatic analysis of the NGS sequencing results.
7. The simplicity of our invention allows maximal flexibility and fast response for emerging new pathogen as well as application at any lab possessing a PCR machine.

Advantages

• HiCRISPR allows fast, high-throughput and accurate determination of spacer sequences of CRISPR-DR regions of any bacteria of interest, resistance genes and other genes of interest.
• Our invention allows fast, cheap and easy profiling of the spacers content in various biological samples (SPACERome).
• Our method allows easy designing of species-specific, or diseases - specific kits and high flexibility.
• We developed a novel and improved barcoding scheme (pro-MID) for easy and individual marking of each sample.
• We developed a novel Python based program (Spopy) for automated data analysis.
• The HiCRISPR method is very easy to operate by any laboratory technician.
• The HiCRISPR method can be run on any 96 wells standard thermocycler.

The HiCRISPR method allows maximal flexibility in designing and fast response to medical and commercial needs. Since our methos is based on sequencing its allow to identify new spacer (in contrast to common methods like spoligotyping) as well as new mutation variants.

The Product

Research and diagnostic tools - Ready to use kits containing all the reagent needed for PCR reaction like polymerase, primers, buffer & nucleotides. The user only need to add water and its DNA samples and to run the PCR machine.

Potential Applications

1. Specific pathogen kit (like Salmonella sp.): typing, detection of resistance & virulence genes.
2. Kits for detection of groups of pathogens: respiratory, intestinal, urinary, etc.
3. SPACERome kit- characterization of complex biological sample by CRISPR/cas DR spacers (relative number of each spacer & DNA sequences).
4. Microbiome applications
5. Environmental applications

The Market

 The global CRISPR and Cas genes market size was valued at USD 1.08 billion in 2015.
 

CRISPR and Cas genes market share, by end-use, 2014 - 2025 (USD Million)

The global microbiology & bacterial culture for industrial (food, water, bioenergy) testing market size was estimated at USD 3.38 billion in 2016. Significant worldwide rise in burden of food-borne illnesses has served as a driving force for the plethora of microbiology researches carried out in recent times. Now with the Microbiom technology , this market will double itself.
 

U.S. microbiology & bacterial culture for industrial testing market, by application, 2014 - 2025
(USD Million)

Field of microbiology research has many key aspects that ensure population safety and product quality. Pure bacterium culture is recognized as an essential tool for investigating virulence factors, antibiotic susceptibility, and genome sequence of microbes to understand their role in infection and food-borne diseases.