Homologous Recombination Reporter Construct for Early Detection
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Solution Overview
Problem
Current methods for detecting homologous recombination events in chromosomal DNA, such as PCR and fluorescent reporters, are inefficient and costly, particularly in zebrafish, where high vivarium costs are incurred from raising large cohorts of animals to biopsy age, and existing reporters are either permanent or not transiently epigenetic, limiting the ability to identify recombination-competent embryos early after injection.
Innovation Solution
Development of homologous recombination reporter constructs that include a mutated fluorescent protein with a sequence element disrupting expression, which is restored upon successful recombination, allowing for early detection of gene-editing events using CRISPR/Cas9 gene editing reagents, enabling the identification of recombination-competent embryos within days of injection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR methods are used to detect homologous recombination events, then detection accuracy is improved, but the requirement to harvest tissue biopsy at advanced age increases vivarium costs and time
Solution Approach 1:
The patent introduces a fluorescent reporter construct that is activated by homologous recombination events, allowing detection to occur early in embryonic development (within days) rather than waiting for biopsy age. The reporter is preliminarily introduced into the embryo along with the gene editing reagents, enabling real-time monitoring of recombination events as they occur during development.
2Loss of time
If fluorescent reporter systems are used to detect gene insertion activity, then early detection capability is improved, but permanent expression or lack of transient epigenetic regulation limits the ability to identify recombination-competent embryos early
Solution Approach 1:
The patent employs a dynamic regulatory system where the fluorescent reporter expression is controlled by the epigenetic state of the integrated transgene. The reporter is silenced in the germline by default (transient epigenetic regulation) but can be activated by demethylating agents or other epigenetic modifiers, allowing flexible temporal control of expression based on experimental needs.
3Reliability
If large cohorts of animals are raised to biopsy age for screening, then detection reliability is improved, but vivarium costs and resource consumption increase significantly
Solution Approach 1:
The patent replaces the mechanical/biological process of raising and biopsying large numbers of animals with an optical detection system. The fluorescent reporter allows non-invasive visualization of recombination events in live embryos using fluorescence microscopy, eliminating the need to maintain large animal cohorts and perform invasive biopsies at advanced stages.
4Loss of time
If existing fluorescent reporters are used, then early detection is improved, but the constructs are either permanent or not transiently epigenetic, limiting flexibility in identifying recombination-competent embryos
Solution Approach 1:
The patent utilizes epigenetic parameters (DNA methylation status, histone modifications) to control reporter expression dynamics. By changing the epigenetic state through chemical treatment or genetic manipulation, the reporter can be switched between silenced and active states, providing temporal and conditional control over fluorescence expression without altering the underlying DNA sequence.
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 HR reporter constructs significantly reduce the number of animals needed to be raised to biopsy age, lowering vivarium costs and increasing the efficiency of detecting successful genome edits by enriching for recombination-competent injections, thereby simplifying the process of isolating germline alleles and reducing screening efforts.
Implementation Method 1
a sequence element that disrupts expression of a functional fluorescent protein and wherein the sequence element is removed with successful homologous recombination in a host cell restoring the functional fluorescent protein
Data Source
AI summary
The present disclosure provides homologous recombination reporter nucleic acid construct reagents for increasing the likelihood of detecting successful modification of a specific sequence in chromosomal DNA of a host cell via homologous recombination. The homologous recombination reporter constructs contain a sequence element inserted within the coding sequence for a reporter gene resulting in a mutated reporter gene. The sequence element is removed via homologous recombination based on the presence of two homology regions present in the reporter construct.