Cell Surface Display Homing Endonucleases for Genomic Toxicity Reduction
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Solution Overview
Problem
Current methods for altering homing endonuclease specificity are limited by the need for intracellular expression and are compromised by genomic toxicity, requiring redesign of cleavage systems for each targeted sequence, and existing screening methods are inefficient and prone to genomic disruption.
Innovation Solution
The method involves cell surface display of homing endonucleases using a recombinant expression system, allowing for high-throughput screening and isolation of variants with novel specificities through flow cytometry and cell separation techniques, enabling the identification of enzymes with altered target site binding and cleavage properties.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If homing endonucleases are expressed intracellularly to alter specificity, then enzyme production is achieved, but genomic toxicity occurs requiring redesign for each target
Solution Approach 1:
The homing endonuclease is extracted from its intracellular environment and displayed on the cell surface, separating the enzyme's catalytic function from the cellular genome. This allows the enzyme to access extracellular DNA targets without causing genomic toxicity, while maintaining the ability to alter specificity for different target sequences.
Solution Approach 2:
The cell membrane serves as an intermediary structure that anchors the homing endonuclease, positioning it to interact with extracellular DNA while preventing direct contact with intracellular genomic DNA. This mediator role eliminates the toxicity problem while preserving the enzyme's DNA-cleaving function.
2Measurement precision
If traditional screening methods are used to identify variants, then selection is possible, but the process is inefficient and prone to genomic disruption
Solution Approach 1:
Traditional mechanical screening methods are replaced with flow cytometry, an optical-based automated system. Fluorescently labeled DNA targets allow rapid, non-contact detection of enzyme-DNA interactions, eliminating genomic disruption risks while dramatically increasing screening throughput and precision.
Solution Approach 2:
Instead of working with the actual genomic DNA targets that risk disruption, fluorescently labeled copies of the target sequences are used. These synthetic DNA copies serve as safe surrogates for screening, allowing accurate variant selection without exposing the cell's genome to cleavage.
3Productivity
If cell surface display is implemented for high-throughput screening, then screening efficiency increases, but system complexity increases
Solution Approach 1:
The cell surface display system uses universal anchoring mechanisms (such as fusion to membrane proteins like CD8 or antibody Fc regions) that can accommodate different homing endonuclease variants. This standardized platform allows high-throughput screening of diverse enzymes without requiring separate complex systems for each variant.
Solution Approach 2:
The homing endonuclease is segmented into functional domains, with the catalytic domain separated from the cell-anchoring domain. This modular architecture allows independent optimization of each component and facilitates high-throughput expression of variant libraries while maintaining manageable system complexity.
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 rapid identification and isolation of homing endonucleases with novel specificities, facilitating targeted DNA strand cleavage and gene manipulation with improved specificity and reduced genomic disruption.
Implementation Method 1
Homing endonucleases (HEs) are highly specific DNA cutting enzymes and recognize DNA target sites ranging from about 14 to about 40 base pairs
Implementation Method 2
assessing the target site binding properties of one or more cell surface displayed functional homing endonucleases... to provide for analysis of their DNA binding by flow cytometry
Data Source
AI summary
According to particular exemplary aspects, DNA target site binding and cleavage properties of native, variant or modified homing endonucleases (HE) (e.g., LAGLIDAG (LHE), HNH, His-Cys Box, GIY-YIG, I-SspI-type, and fusions, muteins or variants thereof) in solution are recapitulated on the cell surface (e.g., as assessed by flow cytometric analysis) to provide for novel cells expressing one or more cell surface HEs (e.g., expressing one or more HE binding and/or cleavage specificities), novel cell libraries, and high-throughput methods for assessing target site binding, target site cleavage. The rapid analysis of HE and LHE-DNA interactions on the cell surface with concurrent sorting options provides for high-throughput library screening affording rapid identification, analysis and isolation of novel HEs or LHEs having novel sequence specificities. Such novel sequence specificities, obtained by said methods provide novel methods for introducing targeted DNA-strand cleavage events, and novel chromatin immunoprecipitation methods (CHIP methods).