Cas9 Gene Copy Multiplication for Multi-Gene Editing
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Solution Overview
Problem
The existing CRISPR system for genetic modification in mice is limited by high levels of Cas9 mRNA, which restricts the amount of guide RNAs and modified genes that can be introduced, and is inefficient for editing multiple genes or locations simultaneously.
Innovation Solution
A genetically modified non-human mammal with a nuclear genome containing at least 3 copies of Cas9 genes, where the Cas9 protein or mRNA accumulates in female reproductive cells, allowing for high-efficiency, site-specific modification of target genes using guide RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high levels of Cas9 mRNA are used in the CRISPR system, then the genetic modification capability is enhanced, but the amount of guide RNAs and modified genes that can be introduced is restricted
Solution Approach 1:
The patent divides the Cas9 delivery system into two separate components: (1) a maternally derived Cas9 protein or mRNA that accumulates in eggs during oogenesis, and (2) paternally derived guide RNAs and modified genes introduced via microinjection. This segmentation allows the Cas9 component to be pre-loaded in large quantities through maternal inheritance, while the guide RNA and modified gene components can be introduced separately without competing for the same injection volume, thereby resolving the contradiction between enhancing genetic modification capability and maintaining the ability to introduce multiple guide RNAs and modified genes.
2Device complexity
If only one copy of Cas9 gene is introduced (as in Cas9 knockin mice), then the genetic modification system is simple, but it is difficult to edit multiple different genes or multiple locations in the same gene simultaneously
Solution Approach 1:
The patent applies preliminary action by pre-accumulating Cas9 protein or mRNA in the egg during oogenesis before fertilization. This preliminary loading of Cas9 ensures that sufficient enzymatic activity is available in advance to support simultaneous or sequential editing of multiple different genes or multiple locations in the same gene, while maintaining system simplicity through maternal inheritance rather than requiring complex multi-copy transgenic constructions.
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 1E
AI summary
The present invention provides a genetic modification non-human organism in which an expression level of Cas9 is high and a plurality of different genes or a plurality of different locations in the same gene can be edited at the same time with high efficiency. The genetic modification non-human organism of the present invention includes a nuclear genome having at least 3 copies of genes that code for Cas9 (CRISPR-associated 9). Egg cells of the present invention are derived from the genetic modification non-human organism having the nuclear genome into which at least 3 copies of genes that code for the Cas9 are introduced. Fertilized eggs of the present invention are obtained by fertilizing the egg cells and sperm derived from the same species of the organism. A method for modifying target genes of the present invention includes a step of introducing a guide RNA into cells derived from the genetic modification non-human organism, the egg cells, or the fertilized eggs.