CRISPR-Edited Non-Human Animal Pluripotent Cells for Antigen-Binding Proteins
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Solution Overview
Problem
Immunization of non-human animals with non-self proteins faces challenges due to immunological tolerance to self-antigens with high sequence homology, and conventional genome editing methods struggle with efficient targeting of certain genomic loci, particularly for large deletions, leading to costly and time-consuming breeding steps to achieve homozygous modifications.
Innovation Solution
A method involving CRISPR/Cas system-guided biallelic modification of genomic loci in non-human animal pluripotent cells to reduce self-antigen expression, followed by immunization with the foreign antigen, resulting in the production of antigen-binding proteins with higher titers and diversity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional immunization methods are used with non-human animals, then the immune system can recognize foreign antigens, but immunological tolerance to self-antigens with high sequence homology prevents effective antibody generation
Solution Approach 1:
The patent applies preliminary anti-action by using CRISPR/Cas9 to preemptively reduce or eliminate self-antigen expression before immunization. By knocking down the self-antigen that causes immunological tolerance, the immune system is prepared in advance to recognize and respond to the foreign antigen without tolerance interference, thereby resolving the contradiction between self-tolerance and foreign antigen recognition
Solution Approach 2:
The patent changes the expression level parameter of the self-antigen gene through CRISPR-mediated genomic modification. By reducing the transcriptional or translational output of the self-antigen, the immune system's tolerance threshold is altered, enabling it to distinguish the foreign antigen more effectively and generate antibodies against epitopes that would otherwise be masked by self-tolerance
2Manufacturing precision
If conventional genome editing methods are used to target genomic loci, then some modifications can be achieved, but large targeted genomic deletions and homozygous modifications are difficult and time-consuming
Solution Approach 1:
The patent applies preliminary action by performing CRISPR/Cas9-mediated genomic modifications on pluripotent stem cells before embryo implantation. This allows biallelic modifications to be established in the germline at the embryonic stage, eliminating the need for multiple generations of breeding that would otherwise be required to achieve homozygous modifications in conventional approaches
Solution Approach 2:
The patent replaces the mechanical breeding process with a molecular biology-based CRISPR editing system. Instead of relying on sequential mating and selection of heterozygous individuals over multiple generations, the Cas9 nuclease guided by specific RNAs directly introduces precise genomic deletions or modifications at the target locus, achieving homozygous changes in a single generation
3Productivity
If self-antigen expression is reduced through genomic modification, then immunological tolerance is decreased and antibody production is enhanced, but the modification process adds complexity to the production method
Solution Approach 1:
The patent uses CRISPR components (Cas9 protein and guide RNAs) as intermediaries to mediate the genomic modification process. These molecular tools enable precise targeting and modification of self-antigen genes without requiring complex experimental procedures, and the modified pluripotent cells serve as intermediaries to generate transgenic animals that consistently produce high titers of antibodies against foreign antigens
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 generates antigen-binding proteins with enhanced affinity and diversity against foreign antigens by reducing self-tolerance and enabling efficient genomic modifications, overcoming limitations of conventional methods.
Implementation Method 1
a first guide RNA that hybridizes to a first guide RNA recognition sequence within a first target genomic locus
Implementation Method 2
The CRISPR/Cas system has provided a new tool for genome editing
Implementation Method 3
introducing the modified non-human animal pluripotent cell into a host embryo
Data Source
AI summary
Methods and compositions are provided for generating antigen-binding proteins against a foreign antigen of interest.