Lambda Beta Protein Mediates Homologous Recombination in Genome Editing
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Solution Overview
Problem
Current genome editing methods in eukaryotic cells, such as the Cre-lox and CRISPR/Cas systems, are time-consuming and inefficient due to issues like off-target effects and low recombination efficiency, making precise and direct genetic modifications challenging, especially in transgenic animals and human cells.
Innovation Solution
A composition and method utilizing Lambda beta protein or a vector encoding Lambda beta protein, combined with capping sequences and potentially exonuclease or anti-RecBCD proteins, to facilitate homologous recombination and precise editing of nucleic acid sequences, including the use of artificial intron sequences with selection markers for efficient gene knockin/knockout and recombineering.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR/Cas system is used for genome editing, then targeting flexibility is improved, but off-target effects increase
Solution Approach 1:
The patent introduces Lambda beta protein as an intermediary component that facilitates homologous recombination between the gRNA-guided Cas complex and the target DNA sequence. This mediator enables more precise recombination by promoting strand invasion and exchange, thereby reducing off-target effects while maintaining targeting flexibility through user-designed gRNA sequences.
Solution Approach 2:
The invention creates a composite genome editing system that combines CRISPR/Cas components (Cas protein, gRNA) with Lambda Red system components (beta protein, capping sequences). This composite approach leverages the targeting flexibility of CRISPR/Cas while incorporating the high recombination precision of Lambda Red, resulting in a system that achieves both adaptability and specificity.
2Productivity
If Cre-lox system is used for gene modification, then recombination efficiency is improved, but device complexity increases
Solution Approach 1:
The patent extracts and utilizes only the essential beta protein component from the Lambda Red system, rather than implementing the complete Cre-lox system with its loxP sequences and Cre recombinase. This extraction approach maintains high recombination efficiency while reducing system complexity by eliminating the need for special lox sequences and complex plasmid constructions.
Solution Approach 2:
The Lambda beta protein component serves multiple functions: it promotes homologous recombination, facilitates strand invasion, and works with various Cas proteins and gRNA designs. This multi-functionality allows the system to achieve high recombination efficiency across different targeting scenarios without requiring separate complex systems for each application.
3Measurement precision
If CRISPR/Cas system is used for genome editing, then targeting precision is improved, but recombination efficiency decreases
Solution Approach 1:
The patent merges the targeting precision mechanism of CRISPR/Cas (gRNA-guided Cas protein complex) with the recombination efficiency mechanism of Lambda Red (beta protein-mediated homologous recombination). By combining these two systems, the invention achieves both high targeting precision through user-designed gRNA and high recombination efficiency through beta protein facilitation of strand invasion and exchange.
Solution Approach 2:
Lambda beta protein acts as an intermediary that bridges the gap between precise CRISPR/Cas targeting and efficient homologous recombination. The beta protein mediates the interaction between the Cas-gRNA complex and target DNA, promoting strand invasion and recombination at the precisely targeted location, thereby combining the strengths of both systems.
4Productivity
If Lambda Red system is used for recombineering, then recombination efficiency is improved, but ease of operation decreases
Solution Approach 1:
The patent segments the Lambda Red system into its essential functional component (beta protein) while eliminating the more operationally complex components (alpha exonuclease, gamma anti-RecBCD, and required capping sequences). This segmentation maintains high recombination efficiency through beta protein-mediated homologous recombination while significantly simplifying the experimental workflow and reducing the number of components that need to be introduced and managed.
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 direct, efficient, and precise editing of target nucleic acid sequences in eukaryotic cells, including human cells, with improved recombination efficiency and reduced off-target effects, facilitating genome modification and gene therapy applications.
Implementation Method 1
Lambda beta protein or a vector comprising a nucleic acid sequence encoding the Lambda beta protein... to facilitate homologous recombination and precise editing of nucleic acid sequences
Data Source
AI summary
Provided is a composition for simultaneously targeting a nucleic acid sequence and providing intron selection in cells in vitro, ex vivo or in vivo. The composition includes one or more nucleic acid molecules each including an artificial nucleic acid sequence flanked with capping sequences, and Lambda beta protein or a linear or circular vector including a nucleic acid sequence encoding the Lambda beta protein, wherein each of the capping sequences is homologous to a region in a target nucleic acid sequence, and the artificial nucleic acid sequence is an intron sequence. The present disclosure also provides a method for editing a target nucleic acid sequence in cells by introducing the composition into the cells.