CRISPR-Cas9 T Cell Gene Editing via Optimized Molar Ratios
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current gene editing methods using the CRISPR/Cas system face challenges in achieving high efficiency, particularly when editing multiple genes simultaneously in T cells, where the efficiency of knocking out specific genes like TCR receptor or HLA protein is limited, especially in terminally differentiated cells like T cells.
Innovation Solution
A method involving a complex of Cas9 enzyme and gRNA is introduced into cells, with a specific molar ratio of 1:3-1:5, where the Cas9 enzyme activity is optimized to 0.1 to 1 nmol, and the complex is used to efficiently edit multiple genes such as TRAC and B2M genes in T cells, achieving high knockout efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR-Cas9 system is used to edit T cells, then gene editing capability is achieved, but gene editing efficiency is low
Solution Approach 1:
The patent optimizes the molar ratio of Cas9 enzyme to gRNA from conventional ratios to a specific range of 1:3 to 1:5, which significantly improves gene editing efficiency. This parameter optimization ensures sufficient gRNA for each Cas9 enzyme while preventing excessive gRNA that could cause non-specific binding, thereby resolving the contradiction between editing capability and editing efficiency
Solution Approach 2:
The patent performs pre-transfection optimization by determining the optimal Cas9:gRNA molar ratio before actual gene editing. This preliminary action includes testing different ratios and selecting the optimal 1:3 to 1:5 ratio, which prepares the system for high-efficiency editing and avoids wasting time and resources on suboptimal conditions
2Adaptability or versatility
If multiple genes are knocked out simultaneously, then comprehensive gene editing is achieved, but editing efficiency decreases
Solution Approach 1:
The patent maintains the optimized Cas9:gRNA molar ratio of 1:3 to 1:5 even when editing multiple genes simultaneously. This consistent parameter optimization ensures that each gRNA has sufficient Cas9 enzyme while preventing competition effects, thereby maintaining high editing efficiency across multiple target genes
Solution Approach 2:
The patent applies the same optimized Cas9:gRNA ratio protocol to multiple different gene targets (TRAC, B2M, and other genes). This universal application of the optimized ratio demonstrates that the 1:3 to 1:5 ratio is broadly applicable across different gene editing scenarios, enabling efficient multi-gene knockout without requiring separate optimization for each gene
3Reliability
If T cells are terminally differentiated, then immune function is maintained, but in vitro amplification time window is limited
Solution Approach 1:
The patent performs gene editing at the optimal time point during T cell culture, before the cells fully differentiate. This preliminary action includes monitoring T cell differentiation status and conducting CRISPR-Cas9 editing when cells are still in a state that allows efficient editing and subsequent amplification, thereby extending the usable time window
Solution Approach 2:
The optimized Cas9:gRNA ratio of 1:3 to 1:5 accelerates the gene editing process, reducing the time required to achieve high knockout efficiency. This faster editing kinetics allows more time for subsequent T cell amplification and expansion, effectively extending the amplification time window while maintaining immune cell function
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 results in a high knockout efficiency of over 90% for the TRAC and B2M genes, enabling rapid and efficient gene editing in T cells, addressing the limitations of existing methods.
Implementation Method 1
RNA-guided Cas endonuclease specifically targets and cleaves DNA in a sequence-dependent manner
Data Source
AI summary
Provided is a method for gene editing of a cell on the basis of a CRISPR/Cas system. The Cas enzyme is a Cas9 enzyme having an enzyme activity of 0.1-1 nmol. Further provided are a method for constructing a universal T cell, a T cell so prepared and use thereof. TCR genes and MHC genes of a T cell are edited by means of gene editing technology. Further provided is a gRNA construct.