Ovarian CRISPR Injection for Faster Germline-Edited Chickens
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Solution Overview
Problem
Current methods for generating genome-edited chickens, such as those using primordial germ cell (PGC)-mediated approaches, are labor-intensive, time-consuming, and pose biosafety risks due to the use of transgenic surrogate hosts, with low germline transmission efficiency and inefficient gene editing.
Innovation Solution
A method involving ovarian injection in situ of CRISPR/Cas9 RNP into a hen's ovarian medulla, followed by crossing with wild-type roosters and detecting gene editing results in the offspring, bypassing PGC-mediated methods to achieve efficient and safe genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PGC-mediated method is used to generate genome-edited chickens, then germline transmission can be achieved, but the process is labor-intensive and time-consuming with low efficiency
Solution Approach 1:
The patent extracts the essential function of PGC-mediated gene editing by directly injecting CRISPR/Cas9 reagents into the ovarian medulla, bypassing the complex PGC isolation, culture, and transplantation process. This extraction of the core editing function while removing unnecessary intermediate steps resolves the contradiction between achieving germline transmission and maintaining high productivity.
Solution Approach 2:
The patent performs preliminary gene editing in the ovarian medulla before egg formation, allowing the CRISPR/Cas9 system to edit both somatic and germline cells simultaneously during follicle development. This preliminary action eliminates the need for subsequent PGC manipulation steps, significantly improving generation efficiency while ensuring germline transmission.
2Reliability
If PGC-mediated method with sterile surrogate chickens is used, then germline transmission efficiency improves, but biosafety risks arise from transgenic surrogate hosts
Solution Approach 1:
The patent removes the transgenic surrogate host component from the gene editing process by directly injecting CRISPR/Cas9 reagents into the ovarian medulla of target hens. This extraction eliminates the biosafety risks associated with transgenic surrogates while preserving the ability to achieve germline transmission through direct editing of the target animal's own germline cells.
3Manufacturing precision
If traditional gene editing methods are used in chickens, then genome modification can be achieved, but the process requires complex PGC culture and transplantation
Solution Approach 1:
The patent merges the editing of somatic cells and germline cells into a single injection procedure by targeting the ovarian medulla, which contains both cell types. This merging eliminates the need for separate PGC isolation, culture, and transplantation steps, significantly reducing experimental complexity while maintaining gene editing precision through direct CRISPR/Cas9 delivery.
4Manufacturing precision
If PGC-mediated gene editing is performed, then genome-edited offspring can be obtained, but at least 18 months are required to obtain homozygous mutants
Solution Approach 1:
The patent performs preliminary gene editing in the ovarian medulla during follicle development, editing both somatic and germline cells simultaneously. This preliminary action creates chimeric G0 chickens that can directly transmit edited genes to G1 offspring, reducing the time to obtain homozygous mutants from 18 months to approximately 8 months by eliminating intermediate breeding generations.
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 allows for the rapid and cost-effective generation of genome-edited chickens with high editing efficiency, enabling identification of mutant individuals within a week and reducing the generation cycle to 8 months, while ensuring biosafety by avoiding exogenous gene introduction.
Implementation Method 1
injecting a gene editing reagent into ovarian medulla of a hen to obtain a G-1 hen; wherein the gene editing reagent includes CRISPR/Cas9 RNP
Data Source
Figure 1A~1D
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AI summary
A method for generating a genome-edited chicken is provided, relating to the technical field of genetic engineering. Ovarian injection in situ is conducted on a hen, where a gene editing reagent is injected into ovarian medulla of the hen that is close to laying eggs, such that the exogenously-injected gene editing reagent can enter developing ovarian follicles through blood circulation. In resulting G0 individuals, a chimera chicken with both somatic cells and germ cells edited is successfully and efficiently obtained, with an editing efficiency of the G0 individuals reaching up to 36.36%. Compared with a traditional primordial germ cell (PGC)-mediated method, the ovarian injection in situ is time-saving and labor-saving, convenient and rapid, low-cost, and highly safe.