CRISPR Knock-in Poultry Egg Ovalbumin Locus
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Solution Overview
Problem
Current methods for expressing proteins in poultry eggs, such as those using lentiviral vectors or retroviral vectors, result in unstable and reduced protein levels due to random gene insertion and silencing effects, making it difficult to maintain high expression levels across generations and in offspring.
Innovation Solution
The development of knock-out poultry eggs with specific oviduct-specific genes eliminated and knock-in eggs with exogenous genes stably expressed under control of oviduct-specific promoters, using CRISPR for precise gene editing to ensure consistent and high expression levels in the oviduct, particularly in the thick albumen.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If gene transfection is performed using lentiviral or retroviral vectors, then protein expression can be achieved, but the expression level varies significantly and becomes unstable across generations
Solution Approach 1:
The patent performs gene knock-in at the ovalbumin locus in primordial germ cells before they differentiate into adult chickens. This preliminary action ensures that the exogenous gene is established in the germline before reproduction, allowing stable inheritance and consistent high-level expression in all offspring without the variability introduced by random viral integration in somatic cells.
Solution Approach 2:
The patent uses the endogenous ovalbumin gene as a template to create a knock-in construct that replicates the natural gene structure and regulation. By inserting the exogenous gene at the ovalbumin locus and using the ovalbumin promoter, the system copies the proven stable expression mechanism of the native gene, ensuring reliable protein production across generations.
2Reliability
If exogenous genes are inserted at random locations in the genome, then gene expression can be achieved, but gene silencing effects reduce expression levels in offspring
Solution Approach 1:
The patent applies local quality by targeting a specific locus (ovalbumin) with known favorable characteristics for gene expression. The ovalbumin locus provides a localized environment with appropriate promoter activity, chromatin structure, and regulatory elements that promote stable expression, avoiding the harmful silencing effects associated with random integration sites.
Solution Approach 2:
The patent uses the endogenous ovalbumin gene sequence as an intermediary framework to host the exogenous gene. The ovalbumin promoter and regulatory elements serve as mediators that facilitate stable expression, while the knock-in construct acts as an intermediary carrier that combines the exogenous coding sequence with the favorable local genomic context.
3Productivity
If viral vectors are used for gene transfection in chicken embryos, then protein expression can be achieved, but the gene insertion is mosaic and cannot be completely transmitted to offspring
Solution Approach 1:
The patent performs gene modification in primordial germ cells before they enter the reproductive lineage. This preliminary action ensures that any genetic changes are established in cells that will become gametes, allowing complete and stable transmission to offspring, unlike somatic cell transfection which creates mosaic patterns that are lost in germline transmission.
Solution Approach 2:
The patent modifies the germline DNA to create a heritable copy of the ovalbumin gene with the exogenous insertion. By working with primordial germ cells that give rise to all gametes, the system creates a uniform genetic copy that is faithfully transmitted to all offspring, eliminating the mosaic variability inherent in viral transfection of embryos.
4Reliability
If the ovalbumin gene is disrupted using TALEN method, then gene knockout can be achieved, but it is difficult to predict future egg production and homozygous knockout feasibility
Solution Approach 1:
The patent uses the well-characterized ovalbumin gene structure as a template for precise knock-in. By inserting the exogenous gene at the ovalbumin locus using CRISPR/Cas9 with designed guide RNAs targeting specific sequences, the system achieves accurate and predictable gene modification, overcoming the uncertainty of TALEN-based knockout approaches.
Solution Approach 2:
The patent changes the gene modification approach from non-specific disruption (TALEN) to precise targeted insertion (CRISPR/Cas9 knock-in). This parameter change in the editing methodology enables predictable outcomes by controlling the exact location and sequence of the insertion, allowing accurate prediction of offspring phenotypes and egg production characteristics.
Data Source
AI summary
Provided are a poultry knock-in egg and knock-out egg. The present invention pertains to a knock-out poultry egg in which at least one oviduct-specific gene has been knocked out, said gene being selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, and lysozyme, and at least one egg allergen protein has been reduced or eliminated, said protein being selected from the group consisting of ovalbumin, ovomucoid, ovomucin, ovotransferrin, ovoinhibitor, and lysozyme.