CRISPR Screening for T Cell Anti-Tumor Response Genes
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Solution Overview
Problem
Conducting subgenome-scale and genome-wide CRISPR/Cas9 screens using human cells is challenging due to difficulties in obtaining monoclonal populations of T cells reactive to tumor antigens, and assessing multiple tumor types with the same population of T cells is nearly impossible.
Innovation Solution
The method involves obtaining T cells or other immune cells engineered with chimeric antigen receptors (CARs) and Cas9, co-culturing them with tumor cells, and using a guide RNA library to selectively edit the cells, allowing for the identification of genes and pathways involved in anti-tumor responses and resistance to tumor suppression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR/Cas9 screens are conducted using human T cells reactive to tumor antigens, then the ability to identify genes and pathways involved in anti-tumor responses is improved, but the difficulty in obtaining monoclonal populations of T cells increases
Solution Approach 1:
The patent uses engineered tumor cells expressing tumor antigens as intermediaries to stimulate and maintain T cell reactivity during CRISPR screening. These engineered tumor cells serve as a mediator that enables the T cells to remain reactive to tumor antigens throughout the screening process, solving the problem of obtaining and maintaining monoclonal T cell populations with specific antigen reactivity.
2Productivity
If the same population of T cells is used to assess multiple tumor types, then the efficiency of screening is improved, but the ability to accurately assess tumor-specific responses deteriorates
Solution Approach 1:
The patent segments the screening system by creating multiple distinct populations of engineered tumor cells, each expressing different tumor antigens. Each T cell population is specifically reactive to one tumor antigen type. This segmentation allows simultaneous assessment of multiple tumor types while maintaining tumor-specific response accuracy, as each T cell population interacts only with its corresponding engineered tumor cell population.
3Quantity of substance
If genome-wide CRISPR/Cas9 screens are performed, then the comprehensiveness of gene identification is improved, but the complexity of the screening system increases
Solution Approach 1:
The patent creates a universal screening platform where engineered tumor cells can be used across multiple CRISPR screens targeting different genes and pathways. The same engineered tumor cell population serves multiple functions: stimulating T cell reactivity, providing a consistent target for screening, and enabling comparison across different genetic perturbations. This universality reduces overall system complexity while maintaining comprehensive gene identification capabilities.
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 the identification of novel genes and pathways mediating immune responses against tumors, facilitating the development of oncology therapies by overcoming previous limitations in screening and editing human cells.
Implementation Method 1
CRISPR site-specific editing in eukaryotic cells has been used for the modification of many plant and animal models. Sternberg and Doudna (2015) Molecular Cell 58: 568-574, describe the development and use of CRISPR-Cas9 for such diverse applications as genome editing, gene regulation, and genome-wide screening systems.
Implementation Method 2
U.S. Pat. No. 8,906,682 describes CAR-T cells which have been engineered to comprise an extracellular domain having an antigen binding domain (such as a domain that binds to CD19), fused to an intracellular signaling domain of the T cell antigen receptor complex zeta chain (such as CD3ξ). When expressed in the T cell, the CAR is able to redirect antigen recognition based on the antigen binding specificity.
Data Source
AI summary
This invention describes a novel CRISPR/Cas9 target identification platform permitting the discovery of novel genes and pathways involved in the ability of T cells and NK cells to react against and generate an anti-tumor response.