Fluorescent Marker Gene for CRISPR Cell Sorting
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Solution Overview
Problem
Current methods for sorting genetically modified cells require an additional analysis step to confirm genetic modification, which is inefficient and results in cell consumption.
Innovation Solution
A method involving fluorescent bovine cells treated with a composition containing guide RNA and Cas protein, allowing for the selection of non-fluorescent cells with modified genes on a target locus without additional analysis, using guide RNA for both the fluorescent protein gene and the target gene.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR analysis is used to confirm genetic modification, then modification detection is achieved, but cells are consumed and additional time is required
Solution Approach 1:
The fluorescent marker gene is introduced into the genome in advance along with the target gene modification. The fluorescent phenotype serves as a preliminary indicator of successful genetic modification, eliminating the need for subsequent PCR analysis and saving time while maintaining detection accuracy.
Solution Approach 2:
Instead of directly analyzing the target gene modification through PCR, a fluorescent marker system is used as a copy or proxy indicator. The presence or absence of fluorescence copying the modification status information, allowing rapid visual detection without consuming cells for molecular analysis.
2Measurement precision
If PCR analysis is used to confirm genetic modification, then modification detection is achieved, but cells are consumed
Solution Approach 1:
The fluorescent marker is established in advance within the cell population. This preliminary action creates a permanent, heritable indicator that allows multiple observations without cell consumption, unlike PCR which requires cell lysis and destroys the sample.
Solution Approach 2:
The fluorescent phenotype serves as a non-consumptive copy of the genetic modification status. Cells can be observed repeatedly under fluorescence microscopy without being consumed, preserving the biological material for further experiments while maintaining accurate detection of modification status.
3Productivity
If fluorescent marker gene is introduced along with target gene, then simultaneous modification is achieved, but device complexity increases
Solution Approach 1:
The fluorescent marker gene and target gene modification are merged into a single genetic construct or delivery system. This combining approach achieves simultaneous modification of both genes in one transfection or transformation event, improving productivity despite the initial complexity of constructing the combined genetic system.
Solution Approach 2:
The fluorescent marker system serves multiple functions: it acts as a selection marker, a visual indicator of successful transfection, and a sorting criterion. This multi-functionality justifies the additional genetic construction complexity by providing comprehensive benefits that simplify downstream processing and increase overall productivity.
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 method enables efficient sorting of genetically modified cells by eliminating the need for additional analysis, preventing cell consumption and reducing time and effort, while allowing for simultaneous modification of both the target locus and fluorescent protein gene.
Implementation Method 1
Recently, widely used genetic modification technologies include Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the CRISPR/Cas system.
Implementation Method 2
preparing fluorescent bovine cells (bovine cells exhibiting fluorescence)
Data Source
AI summary
Provided is a method for more efficiently sorting out genetically modified cells. Specifically provided are a method for selecting a cell including a modified gene on a target locus in a genome, a method for producing a cell including a modified gene on a target locus in a genome, and an animal including a modified gene on a target locus in a genome, and a kit for selecting an animal including a modified gene on a target locus in a genome and cells including a modified gene on a target locus in a genome.