Fully Penetrant Cas/CRISPR Cell Lines for High-Efficiency Genome Editing
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Solution Overview
Problem
Existing genome editing technologies using CRISPR systems are not efficient in achieving high penetrance of RNA-guided endonuclease expression in eukaryotic organisms, leading to suboptimal genome editing outcomes.
Innovation Solution
Development of Eustigmatophyte and Parachlorella microbial strains with fully penetrant expression of Cas/CRISPR systems, utilizing flow cytometry to select cell lines with culture-wide expression of a detectable marker, such as a fluorescent protein, and transforming these strains with guide RNA to achieve high efficiency genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional CRISPR systems are used in eukaryotic organisms, then genome editing can be performed, but the expression penetrance of RNA-guided endonuclease is low leading to suboptimal editing outcomes
Solution Approach 1:
The patent uses a detectable marker (fluorescent protein) as an intermediary to monitor and ensure complete expression of the Cas9 endonuclease. The marker is operably linked to the Cas9 gene, allowing flow cytometry to identify cell populations with full penetrance of endonuclease expression, thereby resolving the contradiction between editing efficiency and expression penetrance
Solution Approach 2:
The patent replaces conventional screening methods with flow cytometry, an optical detection system. By using fluorescent markers that can be detected optically, the system identifies cells with complete Cas9 expression without requiring mechanical manipulation or cultural selection, achieving high productivity and reliability simultaneously
2Reliability
If flow cytometry is used to select cell lines with culture-wide expression of detectable markers, then fully penetrant Cas expression is achieved, but the screening process becomes more complex
Solution Approach 1:
The patent employs fluorescent proteins (color-changing markers) as detectable markers linked to Cas9 expression. Different fluorescence intensities indicate different levels of Cas9 expression, allowing flow cytometry to easily distinguish fully penetrant expressors from partial or non-expressors. This optical signal transformation simplifies the complexity of verifying uniform expression across the culture
3Ease of manufacture
If conventional screening methods are used to identify transformants, then the process is simpler, but the efficiency of genome editing is reduced due to incomplete endonuclease expression
Solution Approach 1:
The patent implements a feedback mechanism where the detectable marker (fluorescent protein) provides real-time information about Cas9 expression status. Flow cytometry measures fluorescence intensity to feedback on which cells have complete Cas9 expression, allowing selection of only those cells for genome editing. This feedback loop ensures high editing efficiency while maintaining relative simplicity in the selection process
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 method results in highly efficient genome editing, with at least 10-95% of cells exhibiting targeted mutations or donor DNA incorporation, surpassing conventional methods by ensuring uniform expression of RNA-guided endonucleases.
Implementation Method 1
Transformed cell lines that include the nucleic acid molecule that includes a gene encoding an RNA-guided nuclease such as a Cas protein and a detectable marker gene are screened by flow cytometry to select a strain or cell line in which essentially all the cells of the culture express the detectable marker, which can be, for example, a fluorescent protein.
Data Source
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AI summary
The present invention provides cell lines for high efficiency genome editing using cas/CRISPR systems, methods of generating such cells lines, and methods of generating mutations in the genome of an organism using such cell lines.