Pathogen Genome Repeat Targeting for CRISPR Resistance Suppression
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Solution Overview
Problem
Antimicrobial resistance in pathogens has become a significant challenge due to the development of resistance mechanisms, such as biofilm formation and mutation of key proteins, rendering conventional treatment strategies ineffective.
Innovation Solution
A system and method for identifying candidate target sites in pathogenic genomes by sequencing DNA from infected samples, recognizing nucleotide repeat sequences that occur multiple times and are dispersed across the genome, and administering an engineered polynucleotide construct to cleave these sequences and flanking genes, using enzymes like CRISPR-Cas to disrupt pathogen functions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional antibiotics are used to treat pathogenic infections, then the treatment is effective initially, but the pathogens develop antibiotic resistance over time
Solution Approach 1:
The CRISPR-Cas system segments the pathogen genome into multiple targetable repeat sequences scattered throughout the genome. By targeting multiple dispersed locations simultaneously rather than a single site, the system creates multiple independent failure points for resistance development, thereby maintaining treatment effectiveness over time.
Solution Approach 2:
The engineered polynucleotide construct serves multiple functions: it delivers guide RNA sequences that target specific repeat regions, recruits Cas enzymes for cleavage, and coordinates the action of multiple enzymes (Cas9, Cas12a, exonuclease) to achieve both genome cleavage and flanking gene removal. This multi-functional approach addresses both the need for effective treatment and prevention of resistance.
2Device complexity
If single-target therapeutic strategies are used against pathogens, then the treatment mechanism is simple, but the pathogens can easily mutate and evade the treatment
Solution Approach 1:
The treatment mechanism is segmented into multiple independent components: multiple guide RNA sequences targeting different repeat regions, multiple Cas enzymes with different PAM requirements, and auxiliary enzymes for flanking gene removal. This segmentation ensures that a single mutation cannot evade all targets simultaneously.
Solution Approach 2:
The therapeutic approach uses a composite strategy combining CRISPR-Cas9, CRISPR-Cas12a, and exonuclease enzymes working together. Each enzyme has distinct properties and target requirements, creating a composite system where the failure of one component does not compromise the overall treatment effectiveness.
3Reliability
If biofilm inhibitors are used to prevent biofilm formation, then biofilm formation is reduced, but pathogens develop resistance through efflux pumps
Solution Approach 1:
The system extracts and targets the underlying genetic elements (repeat sequences and flanking genes) that control biofilm formation and resistance mechanisms. By removing these genetic determinants through CRISPR-Cas mediated cleavage and exonuclease activity, the system addresses the root cause rather than just the symptomatic biofilm structure.
4Reliability
If multiple target sites are targeted simultaneously in the pathogen genome, then the likelihood of resistance development is reduced, but the complexity of the treatment system increases
Solution Approach 1:
Multiple targeting functions are merged into a single engineered polynucleotide construct that delivers multiple guide RNA sequences and recruits multiple Cas enzymes simultaneously. This merging approach achieves multi-targeting without proportionally increasing system complexity, as all components are coordinated through a single construct delivery mechanism.
Solution Approach 2:
The CRISPR-Cas system is designed to be self-guiding, where the guide RNA sequences within the construct automatically direct the Cas enzymes to their complementary target sites in the pathogen genome. This self-service mechanism reduces the need for complex external control systems, allowing multiple targets to be addressed simultaneously with minimal additional complexity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach effectively debilitates multiple critical pathogen functions simultaneously, making it difficult for pathogens to develop resistance, thereby providing a robust treatment strategy.
Implementation Method 1
a first enzyme capable of nicking and cleaving the identified set of nucleotide sequences
Implementation Method 2
a second enzyme capable of removal of a set of neighborhood genes flanking the set of nucleotide repeat sequences
Implementation Method 3
The constructed polynucleotide comprises a set of guide sequences, a first enzyme capable of nicking and cleaving the identified set of nucleotide sequences
Data Source
AI summary
A system and method for identification of target sites in a pathogenic genome and combating a pathogenic infection have been provided. The present disclosure utilizes the fact that a conserved stretch of nucleotide sequence in genomic neighborhood of genes important for bacteria can be targeted to disrupt the overall functioning of the pathogen. The method involves identification of nucleotide repeat sequences in the DNA. The method and system also involve administration of a cocktail comprising antimicrobial drugs, biofilm inhibitors and a construct. The genomic neighborhood or vicinity or ‘flanking genes’ refers to regions lying within a predefined number of genes to the identified conserved stretch of nucleotide repeat sequence (or its reverse complement) on the candidate pathogen genome or within a distance of predefined number of bases with respect to the conserved stretch of nucleotide repeat sequence (or its reverse complement) on the candidate pathogen genome.