FAME Method for Double Knockout Cell Enrichment
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Solution Overview
Problem
Current gene editing techniques, particularly CRISPR, face inefficiencies in achieving double knockout mutations in diploid eukaryotic cells, requiring extensive manual labor and time for single cell colony isolation and genotyping, with low success rates in enriching for desired mutations.
Innovation Solution
The Flow Assisted Mutation Enrichment (FAME) method uses FACS-based negative selection to enrich for cells with double knockout mutations by targeting and ablating a surface marker gene, such as B2M, allowing for automated single cell isolation and significantly increasing the prevalence of indel mutations to nearly 100% in negatively selected cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If manual single cell colony isolation and genotyping is used, then genotyping accuracy is maintained, but productivity is low and time consumption is high
Solution Approach 1:
The patent replaces manual mechanical isolation and genotyping operations with automated FACS-based cell sorting and high-throughput sequencing. The FACS system automatically isolates single cells based on fluorescence markers, and NGS automatically genotypes multiple colonies in parallel, substituting labor-intensive manual processes with automated mechanical and computational systems.
Solution Approach 2:
The patent introduces a preliminary fluorescent marker system that labels cells with desired mutations before isolation. By pre-marking target cells with fluorescent proteins or dyes, the system enables automated FACS to quickly identify and sort desired cells without time-consuming manual screening, performing the selection function in advance of the isolation step.
2Reliability
If CRISPR editing is applied to diploid eukaryotic cells, then gene editing efficacy is improved, but the success rate of achieving double knockout mutations decreases
Solution Approach 1:
The patent uses fluorescent markers as intermediary indicators to track and identify cells with desired double knockout mutations. By linking fluorescent marker expression to the successful occurrence of dual mutations, the system provides a visual and quantifiable intermediary signal that guides FACS sorting, making the selection of DKO cells highly efficient without requiring screening of numerous colonies.
Solution Approach 2:
The patent implements a feedback mechanism where fluorescent marker expression provides real-time information about the success of CRISPR editing. Cells exhibiting the desired fluorescence pattern immediately feedback their mutant status, enabling automated sorting systems to selectively enrich DKO cells while discarding non-target cells, thereby dramatically improving enrichment efficiency without increasing colony screening numbers.
3Measurement precision
If extensive manual labor is used for colony isolation and genotyping, then genotyping precision is maintained, but device complexity increases
Solution Approach 1:
The patent employs universal fluorescent marker systems and standardized FACS protocols that can be applied across different cell types and CRISPR target sites. The same basic workflow—transfection with CRISPR components plus fluorescent marker, FACS sorting based on fluorescence, and NGS validation—can universally handle diverse gene editing experiments, reducing the need for complex, specialized procedures for each specific application.
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
FAME effectively enriches for double knockout mutations in target genes, reducing the time and labor required, achieving unprecedented success rates in CRISPR gene editing by automating the isolation and verification of cells with desired mutations.
Implementation Method 1
performing FACS-based negative selection to establish an enriched cell population of negatively selected cells
Data Source
AI summary
Disclosed are methods comprising administering CRISPR technology to a population of cells, wherein the CRISPR technology comprises one or more constructs for expressing a Cas protein, sgRNA against a marker gene, and sgRNA against a target sequence; and performing FACS-based negative selection to establish an enriched cell population of negatively selected cells; wherein the negatively selected cells do not have a marker encoded by the marker gene and do have a mutation in the target sequence. Disclosed are nucleic acid sequences comprising three elements, wherein a first element comprises a nucleic acid sequence that encodes a Cas protein, a second element comprises a nucleic acid sequence that expresses a sgRNA against a cell-surface marker gene, and a third element comprising a nucleic acid sequence that expresses a sgRNA against a target sequence.