Primate Liver Tumor Model via Hepatic Portal CRISPR Injection
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Solution Overview
Problem
The construction of primate disease models with precisely modified specific genes is challenging due to the immaturity of embryonic stem cell and somatic cell nuclear transplantation technologies in non-human primates, resulting in long cycles and high costs, and existing CRISPR technology is not effective for somatic cell-level modifications in cynomolgus monkeys.
Innovation Solution
A method involving the construction of a sgRNA expression plasmid using a gRNA oligonucleotide and pX330 plasmid, followed by direct injection into the hepatic portal vein of primate animals to induce gene mutations in liver cells, specifically targeting the p53 gene using the CRISPR-Cas9 system for rapid tumor model creation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional gene targeting technology based on embryonic stem cells or somatic cell nuclear transplantation is used, then precise gene modification can be achieved, but the construction cycle is long and cost is high
Solution Approach 1:
The patent extracts the CRISPR-Cas9 gene editing system from traditional embryonic stem cell or somatic cell nuclear transplantation methods. By directly injecting the sgRNA expression plasmid into the hepatic portal vein, the system bypasses the need for complex transplantation procedures, thereby maintaining gene modification precision while dramatically reducing the construction cycle and cost.
Solution Approach 2:
The patent uses the hepatic portal vein as an intermediary pathway to deliver the CRISPR-Cas9 system directly to liver cells. This intermediary route enables efficient gene delivery without requiring embryonic stem cell manipulation or somatic cell nuclear transplantation, thus achieving precise gene modification in a much shorter time frame.
2Manufacturing precision
If traditional gene targeting technology is used, then precise gene modification can be achieved, but the construction cost is high
Solution Approach 1:
The patent extracts the essential gene editing function from expensive traditional methods and implements it through a simpler, more cost-effective CRISPR-Cas9 system delivered via hepatic portal vein injection. This approach maintains precise gene modification capability while significantly reducing the quantity of resources required and overall construction cost.
Solution Approach 2:
The patent employs a disposable sgRNA expression plasmid that can be directly injected and functions transiently to achieve the desired gene modification. This eliminates the need for expensive, long-term maintenance of embryonic stem cell lines or somatic cell cultures, thereby reducing construction costs while maintaining precision.
3Productivity
If CRISPR technology is applied at somatic cell level in cynomolgus monkeys, then rapid disease model construction is possible, but traditional methods show it is not available
Solution Approach 1:
The patent uses the hepatic portal vein as an intermediary delivery system to make CRISPR technology available for somatic cell-level modification in cynomolgus monkeys. This intermediary route overcomes the technical barriers that previously made CRISPR inapplicable at the somatic cell level in these animals, enabling rapid and reliable disease model construction.
Solution Approach 2:
The patent changes the delivery parameters by moving from traditional in vitro or germline approaches to in vivo direct injection via the hepatic portal vein. This parameter change makes CRISPR technology available and effective for somatic cell modification in cynomolgus monkeys, achieving both high productivity and reliability in disease model construction.
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 method enables the rapid and efficient construction of primate disease models by directly injecting the CRISPR-Cas9 system into the liver, achieving gene mutations and inducing liver cancer, thereby overcoming the limitations of traditional methods and demonstrating a high mutation rate and successful tumor model induction.
Implementation Method 1
CRISPR-RNA directs the CAS protein to perform a specific splicing and generates double strand breaks (DSBs) at the DNA target sites
Implementation Method 2
injecting the sgRNA expression plasmid prepared in step (a) into a hepatic portal vein of a primate animal by using a biopsy needle
Implementation Method 3
DSB, which is generated after the DNA damage, repairs the damaged DNA for editing the genome at the fixed point, through activating two different intracellular intrinsic repair mechanisms of NHEJ (Non-homologous ending-joining) and HR (Homologous recombination)
Implementation Method 4
DSB, which is generated after the DNA damage, repairs the damaged DNA for editing the genome at the fixed point, through activating two different intracellular intrinsic repair mechanisms of NHEJ (Non-homologous ending-joining) and HR (Homologous recombination)
Data Source
AI summary
Provided is a primate disease model construction method based on fast gene edition, which including (a) constructing a sgRNA expression plasmid by using a gRNA oligonucleotide and a pX330 plasmid; (b) injecting the sgRNA expression plasmid prepared in step (a) into a hepatic portal vein of a primate animal by using a biopsy needle until liver cells become cancerous for obtaining a primate disease model. The sgRNA expression plasmid constructed by the gRNA oligonucleotide and pX330 plasmid can be directly injected into the primate liver tissue, so as to construct a tumor model rapidly.