Lytic Bacteriophage CRISPR Targeting Essential Genes
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Solution Overview
Problem
Current bacteriophage therapies are limited in their ability to effectively target and kill multi-drug resistant bacteria, particularly Clostridium difficile, due to their temperate nature, which allows them to enter a lysogenic state and evade lytic replication, making them ineffective against essential bacterial genes.
Innovation Solution
Engineering bacteriophages to be lytic by removing or inactivating lysogenic genes, such as the cI repressor region, and incorporating CRISPR arrays that target essential bacterial genes, ensuring the bacteriophages remain lytic and effectively kill target bacteria.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If temperate bacteriophages are used for therapy, then they can infect bacteria, but they enter lysogenic state and evade lytic replication making them ineffective
Solution Approach 1:
The patent removes or inactivates the lysogenic gene (such as the cI repressor region) from the temperate bacteriophage genome. This extraction of the lysogenic capability forces the bacteriophage to maintain a lytic lifecycle, ensuring reliable therapeutic efficacy by preventing the phage from entering a dormant lysogenic state that would evade lytic replication and bacterial killing.
2Productivity
If CRISPR arrays target essential bacterial genes, then bacteriophages can kill target bacteria effectively, but it requires precise targeting of specific genetic sequences
Solution Approach 1:
The patent incorporates pre-designed CRISPR arrays with specific spacer sequences into the bacteriophage genome before infection. These CRISPR arrays are engineered to target essential bacterial genes, and the bacteriophage delivers them directly into the target bacterium during infection. The CRISPR-Cas system then uses these pre-programmed guides to precisely locate and cleave the target bacterial DNA, achieving high bacterial killing efficiency through precise genetic targeting.
3Stability of the object's composition
If bacteriophages are engineered to be lytic by removing lysogenic genes, then they maintain consistent lytic activity, but the genetic modification increases device complexity
Solution Approach 1:
The patent converts the potential harm of genetic modification (increased complexity) into a benefit by strategically removing only the critical lysogenic gene elements (such as the cI repressor region) while preserving the rest of the bacteriophage genome. This targeted removal approach achieves stable lytic activity with minimal genetic changes, reducing the overall complexity burden while ensuring the phage maintains consistent lytic behavior for reliable therapy.
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
The engineered lytic bacteriophages efficiently kill target bacteria by maintaining lytic activity and targeting essential genes, even in the presence of antibiotic resistance, thereby addressing the limitations of temperate bacteriophages and enhancing therapeutic efficacy against drug-resistant strains.
Implementation Method 1
a bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium
Implementation Method 2
the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium
Implementation Method 3
provided that the bacteriophage is rendered lytic
Implementation Method 4
the bacteriophage infects multiple bacterial strains
Data Source
AI summary
Disclosed herein are compositions and methods for modifying a bacterial population. In some embodiments, described herein is a bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic.