Type I-F3 CRISPR-Transposon Integration for Specific Bacterial Editing
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Solution Overview
Problem
Existing CRISPR-Cas systems for DNA integration are limited in their efficiency and specificity, particularly in the context of RNA-guided DNA integration using Tn7-like transposon systems, which lack comprehensive integration capabilities across diverse bacterial species.
Innovation Solution
Development of engineered CRISPR-Cas systems combined with Tn7-like transposon systems derived from various bacterial species, including Aliiglaciecola sp., Vibrio sp., Halomonas titanicae, Photobacterium aquae, Photobacterium iliopiscarium, Photobacterium piscicola, Psychromonas sp., Klebsiella oxytoca, Colwellia polaris, and Neptunomonas qingdaonensis, to enhance RNA-guided DNA integration and modification capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If existing CRISPR-Cas systems are used for DNA integration, then genome editing capability is provided, but integration efficiency and specificity are limited
Solution Approach 1:
The patent combines CRISPR-Cas systems with Tn7-like transposon systems into a hybrid system. The CRISPR-Cas component provides target site recognition through guide RNA, while the Tn7-like transposon component (including TnsA, TnsB, TnsC, and TniQ proteins) provides efficient and specific DNA integration capability. This merging resolves the contradiction by integrating the targeting precision of CRISPR with the integration efficiency of transposons.
Solution Approach 2:
The invention creates a composite molecular system that integrates two different biological mechanisms: the RNA-guided nuclease system of CRISPR-Cas and the transposase-mediated DNA transfer system of Tn7-like transposons. This composite system achieves both high integration efficiency from the transposon machinery and high specificity from the CRISPR guide RNA-target recognition.
2Adaptability or versatility
If CRISPR-Cas systems are used alone, then adaptive immune function is provided, but DNA integration capability is insufficient
Solution Approach 1:
The patent merges the adaptive immune/targeting function of CRISPR-Cas with the DNA integration function of Tn7-like transposons. The CRISPR array and Cas proteins provide target recognition and binding, while the transposon proteins (TnsA, TnsB, TnsC, TniQ) provide the molecular machinery for efficient DNA integration, thereby achieving both versatility and productivity.
Solution Approach 2:
The hybrid system achieves multi-functionality by combining the targeting versatility of CRISPR (which can be programmed to recognize any DNA sequence with appropriate PAM) with the integration efficiency of transposons. This allows the system to perform both immune-like targeting and efficient genome editing functions.
3Productivity
If Tn7-like transposon systems are used alone, then DNA integration is provided, but target site specificity is limited
Solution Approach 1:
The CRISPR guide RNA acts as an intermediary that bridges the transposon integration machinery and the target DNA sequence. The guide RNA recognizes and binds to the specific target site through complementary base pairing, directing the Tns proteins to the correct location for integration, thereby providing target site specificity to the otherwise non-specific transposon system.
Solution Approach 2:
The system segments the DNA integration process into two distinct functions: target recognition (performed by CRISPR guide RNA-Cas complex) and integration execution (performed by Tns proteins). This segmentation allows each component to optimize its specific function while working together as a unified system.
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 systems demonstrate improved integration efficiency and specificity, enabling targeted DNA integration, genome modification, gene editing, and cell killing, as well as screening for non-essential genes and altering gene expression in diverse bacterial species.
Implementation Method 1
a guide nucleic acid comprising at least one spacer having complementarity to a target site present in a nucleic acid present in the cell
Implementation Method 2
an engineered transposon 7-like (Tn7-like) transposon system comprising: (a) Transposon 7 protein A (TnsA), (b) Transposon 7 protein B (TnsB), (c) Transposon 7 protein C (TnsC), and (d) transposition of integron protein Q (TniQ)
Data Source
AI summary
This invention relates to recombinant nucleic acid constructs encoding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems and transposon 7-like (Tn7-like) transposon systems for DNA integration, as well as methods of using the same.