Cms1 CRISPR System for Selective Bacterial Elimination
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for selectively eliminating pathogenic bacteria are hindered by antibiotic resistance and inefficiencies in CRISPR systems, such as Type I CRISPR systems being large and multi-component, and Cas9 nucleases causing bacteria to survive due to single DSB repair mechanisms.
Innovation Solution
The use of Cms1 CRISPR systems, which include DNA constructs encoding Cms1 proteins operably linked to promoters and guide RNAs, allowing for sequence-specific targeting and elimination of bacterial cells by introducing double-stranded breaks and subsequent degradation of DNA or RNA, without the need for additional trans-activating crRNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Type I CRISPR systems are used to selectively eliminate bacteria, then bacterial elimination capability is improved, but system size and complexity increase making packaging difficult
Solution Approach 1:
The patent extracts and utilizes only the essential Cas12a nuclease component and guide RNA from the Type V CRISPR system, eliminating the need for multiple additional proteins and complex machinery required by Type I systems. This extraction of core functional elements achieves bacterial elimination while maintaining system compactness for easy packaging.
Solution Approach 2:
The Cas12a nuclease performs multiple functions: it binds to guide RNA for sequence-specific targeting, cleaves double-stranded DNA at target sites, and exhibits collateral cleavage activity on non-specific dsDNA. This multi-functionality consolidates what would require multiple separate components in Type I systems into a single versatile enzyme.
2Measurement precision
If Cas9 nucleases are used to target bacteria, then sequence-specific targeting is achieved, but bacteria survive due to single DSB repair mechanisms
Solution Approach 1:
The patent exploits the collateral cleavage activity of Cas12a as a beneficial feature. After Cas12a binds to its specific target sequence, it undergoes a conformational change that enables it to non-specifically cleave other dsDNA molecules in trans. This collateral activity converts the enzyme's activated state into a lethal event for the bacterial cell, ensuring elimination even if the primary target repair succeeds.
Solution Approach 2:
Once Cas12a is activated by binding to its target sequence, it continuously cleaves dsDNA molecules in a trans manner until depleted. This continuous destructive action ensures that even if some bacteria repair the primary DSB, the ongoing collateral cleavage activity prevents survival, maintaining reliable elimination throughout the bacterial population.
3Productivity
If traditional bacterial control methods using antibiotics are used, then bacterial population reduction is achieved, but antibiotic resistant bacteria emerge as health threats
Solution Approach 1:
The patent replaces the chemical mechanism of antibiotics with a biological molecular recognition system. CRISPR-Cas12a uses guide RNA to specifically recognize and bind to complementary bacterial DNA sequences through base pairing, then cleaves the target DNA. This sequence-specific molecular mechanism eliminates bacteria without applying selective chemical pressure that drives antibiotic resistance development.
Solution Approach 2:
The CRISPR system applies elimination pressure locally and specifically to bacteria containing the target sequence, rather than universally to all bacteria. The guide RNA confers sequence-specificity, so only bacteria with the matching DNA sequence are targeted and eliminated. This localized action prevents the widespread selective pressure that leads to resistance development across bacterial populations.
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
Cms1 CRISPR systems enable selective killing of bacterial cells harboring specific DNA sequences while sparing cells without the target sequence, offering improved specificity and efficiency over existing CRISPR systems.
Implementation Method 1
the Cms1 polypeptide hybridizes with a targeted sequence in one or more bacterial cells of interest and cleaves the targeted sequence
Implementation Method 2
The Cms1 polypeptide may be a CRISPR-associated endonuclease
Implementation Method 3
the guide polynucleotide is designed to interact, and capable of interacting, with a Cms1 polypeptide and to hybridize with a targeted sequence
Data Source
AI summary
Compositions and methods for targeting pre-determined DNA sequences in bacterial cells are provided. The methods result in the targeted elimination of bacterial cells that comprise the pre-determined DNA sequence(s). Compositions comprise DNA constructs comprising nucleotide sequences that encode a Cms1 protein operably linked to a promoter that is operable in the cells of interest. Methods to use these DNA constructs to selectively target and eliminate bacterial cells that harbor the targeted DNA sequence(s) are described herein.