CRISPR Screening System for Mutation Analysis
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for assessing the effects of genetic sequence variants on phenotypic parameters of cells are laborious, time-consuming, and costly, often resulting in false positive results due to factors like transfection toxicities and off-target effects, and require lengthy clone generation, limiting their clinical and therapeutic applications.
Innovation Solution
A method involving the introduction of a mutation of interest and a synonymous mutation into a cell population using a nuclease, allowing for the simultaneous analysis of hundreds of knock-in cells, which eliminates the need for clone generation and provides a fast, reliable assessment of phenotypic effects through PCR or next-generation sequencing, using internal controls to validate results.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional knock-in experiments using gene-editing technologies are performed with clonal cell population generation, then the functional consequence of mutations can be assessed, but the process is laborious, time-consuming, and expensive with high propensity for false positive results
Solution Approach 1:
The patent segments the cell population into multiple independent cultures, each receiving different oligonucleotide combinations (mutation of interest vs synonymous control). This allows parallel assessment of multiple conditions without requiring sequential clone generation, thereby reducing time while maintaining reliability through statistical comparison across segments
Solution Approach 2:
The patent introduces synonymous mutations as intermediary control elements that serve as internal references within the same experimental system. These control mutations allow normalization of results to account for experimental variables like transfection efficiency and off-target effects, thereby improving reliability without extending the time required for analysis
2Reliability
If conventional methods with multiple repeat experiments and individually designed controls are used, then false positive results can be reduced, but labor and material costs increase significantly
Solution Approach 1:
The patent merges the mutation of interest and synonymous control into a single co-transfection experiment using pooled oligonucleotides. This combines multiple control functions (transfection efficiency, off-target effects, background variability) into one integrated experimental unit, reducing the need for separate repeat experiments and thereby improving cost efficiency while maintaining reliability
Solution Approach 2:
The synonymous mutation serves multiple functions simultaneously: it acts as a control for transfection efficiency, a reference for off-target effect normalization, and a baseline for comparing phenotypic changes. This multi-functionality eliminates the need for multiple specialized controls, reducing both labor and material costs while maintaining experimental reliability
3Measurement precision
If clonal cell lines are generated for mutation analysis, then individual mutation effects can be determined, but the process is inherently lengthy and requires that the mutation allows clone expansion
Solution Approach 1:
The patent uses oligonucleotide copies of the desired mutation sequences to introduce changes directly into the genome without requiring physical clone isolation and expansion. By using synthetic oligonucleotides as templates for homology-directed repair, the method bypasses the complex multi-step clone generation process while maintaining precision in determining mutation effects through sequence-specific analysis
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 accurate, fast, and cost-efficient determination of the effects of genetic mutations on cell parameters like proliferation and survival, reducing the time to results from weeks to a few weeks and eliminating the need for lengthy clone generation, while minimizing false positives.
Implementation Method 1
a nuclease or a polynucleotide encoding said nuclease, wherein the nuclease is capable of generating one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) in a target nucleic acid sequence
Implementation Method 2
determining the effect of the mutation of interest on the parameter of interest... allowing absolute frequencies to be calculated
Implementation Method 3
In one design of the PCR or next-generation sequencing (NGS) analysis of the introduced mutations, primers are designed to anneal outside the region substantially similar to the oligonucleotides introduced, allowing absolute frequencies to be calculated
Data Source
AI summary
The present disclosure relates to methods for assessing the effects of a mutation of interest in a cell. Herein are also disclosed systems for assessing the effects of a mutation of interest in a cell. The disclosure also provides host cells and host cell populations comprising the system.