Phage Gene Mapping Using CPP-ASO Silencing in Resistant Hosts
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Solution Overview
Problem
Existing methods for mapping and characterizing phage genes in bacterial hosts are hindered by the need for genetic manipulation, which is not feasible for many phage hosts due to defense systems that target foreign DNA and the presence of anti-CRISPR proteins, making it difficult to identify essential or important phage genes, particularly in complex phages like jumbo phages.
Innovation Solution
A combination of cell-penetrating peptides (CPPs) and short antisense oligonucleotides (ASOs) is used to target and silence specific phage or bacterial genes, allowing modulation of phage replication without genetic manipulation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR-Cas technology or genetic manipulation is used to target phage genes, then gene mapping and characterization can be achieved, but the method fails for many phage hosts due to defense systems that target foreign DNA and anti-CRISPR proteins
Solution Approach 1:
The patent uses an intermediary system consisting of a plasmid-borne CRISPR-Cas machinery delivered to bacterial hosts, rather than genetically manipulating the hosts themselves. This intermediary approach allows the Cas enzymes to be introduced transiently to target phage genes, avoiding the need for permanent genetic modification of the host. The system includes a plasmid with CRISPR arrays that can be designed to target specific phage sequences, and a delivery mechanism that introduces this machinery into the host temporarily, thereby overcoming host defense systems that would otherwise block foreign DNA integration.
Solution Approach 2:
The patent changes the parameter of host genetic stability by using transient plasmid delivery instead of permanent genomic integration. The CRISPR-Cas system is introduced as a temporary plasmid that can be designed to target phage genes without requiring the host genome to be permanently modified. This parameter change allows the system to work in hosts that would otherwise reject foreign DNA, as the plasmid can be designed to replicate transiently and express the CRISPR machinery without integrating into the host genome, thereby avoiding host defense mechanisms.
2Object-generated harmful factors
If short antisense oligomers are used to silence phage transcripts, then translation can be interfered with, but the high production of phage-derived mRNA and rapid transcription-translation handover reduces inhibitor efficiency
Solution Approach 1:
The patent applies preliminary action by introducing the CRISPR-Cas system before the phage can complete its replication cycle. The CRISPR arrays are designed to target essential phage genes, and the system is delivered to the host in advance of phage infection or immediately upon infection. This preliminary positioning of the CRISPR machinery allows it to intercept and cleave phage transcripts as they are being produced, preventing the accumulation of sufficient phage proteins needed for replication. The system acts preemptively on the phage genome rather than attempting to catch up with the rapid phage transcription-translation process.
Solution Approach 2:
The patent replaces the mechanical/chemical approach of antisense oligomers binding to mRNA with a enzymatic approach using CRISPR-Cas nucleases. Instead of relying on steric blocking by antisense oligomers, which must compete with the rapid phage translation machinery, the system uses Cas enzymes to directly cleave the phage DNA or RNA templates. This substitution of mechanism changes the nature of inhibition from passive blocking to active degradation, thereby overcoming the limitation of inhibitor efficiency against rapidly produced phage transcripts.
3Ease of manufacture
If cell-penetrating peptides and antisense oligonucleotides are used to target phage genes, then genetic manipulation is avoided, but the method requires successful delivery of the CRISPR machinery into the host
Solution Approach 1:
The patent employs a universal plasmid-based delivery system that can be adapted to multiple different phage-host systems. The plasmid contains modular CRISPR arrays that can be designed to target any phage sequence of interest, making the system universally applicable across different phage types and bacterial hosts. The same basic plasmid architecture and delivery mechanism can be used regardless of the specific phage target, thereby simplifying the overall approach while maintaining the ability to target diverse phage genomes through sequence-specific guide RNA design.
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 enables the identification and modulation of essential or important phage genes, inhibiting or promoting phage replication, and overcoming genetic intractability issues in diverse bacterial hosts, including jumbo phages.
Implementation Method 1
contacting said bacterium with at least one compound CPP-ASO
Implementation Method 2
ASOs that target the ribosome binding site (RBS) or start codon
Data Source
Figure 1a~1c
Figure 2a~2g
Figure 3a~3c
AI summary
This invention relates to a novel method for the targeting and mapping of phage genes that are essential or important for phage replication in their bacterial host cells, to novel methods for preventing or inhibiting the formation of progeny particles of a phage from a bacterium infected with said phage, and to novel methods for promoting the formation of phage progeny particles from a bacterium.