Transgenic Maize Gene Excision via Site-Specific Nucleases
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Solution Overview
Problem
Transgenic maize plants often contain undesirable rearrangements and selectable marker genes that are no longer needed, posing challenges for efficient trait expression and genetic modification.
Innovation Solution
The development of transgenic maize plant cells with specific genetic constructs, including the ZmUbiInt promoter, vip3Aa19 coding region, and nopaline synthase terminator elements, which allow for the removal of phosphomannose isomerase selectable marker genes and repetitive sequences, using site-specific nucleases and guide RNAs to excise unwanted DNA elements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If transgenes are integrated into the plant genome through non-site specific integration, then transgene expression can be achieved, but undesirable rearrangements and selectable marker genes are introduced
Solution Approach 1:
The patent applies the extraction principle by removing unwanted DNA elements (selectable marker genes and repetitive sequences) from the transgene insertion site while retaining the functional transgene. This is achieved through site-specific nuclelease treatment that targets and excises specific DNA sequences flanking the transgene, thereby eliminating harmful factors without compromising transgene expression reliability.
Solution Approach 2:
The patent segments the transgene expression cassette from surrounding DNA elements by defining precise boundaries using site-specific nuclelease recognition sites. This segmentation allows independent manipulation of the transgene core function while removing flanking unwanted sequences, resolving the contradiction between maintaining expression and eliminating harmful rearrangements.
2Ease of manufacture
If selectable marker genes are retained in transgenic plants, then trait selection can be performed, but genetic stability and purity are compromised
Solution Approach 1:
The patent extracts and removes selectable marker genes from the transgenic plant genome after the desired trait has been selected. This is accomplished by using site-specific nucleases to recognize and excise DNA sequences containing the marker genes, thereby achieving genetic stability and purity while maintaining the ability to perform trait selection during the breeding process.
Solution Approach 2:
The patent performs preliminary selection using selectable marker genes during the breeding process, then subsequently removes the markers through site-specific nuclelease treatment. This preliminary action allows easy trait selection followed by genetic cleanup, resolving the contradiction between selection capability and genetic stability.
3Productivity
If multiple DNA elements are present at transgene insertion sites, then initial transformation can be achieved, but genetic complexity and unwanted traits increase
Solution Approach 1:
The patent extracts and removes repetitive sequences and extraneous DNA elements from the transgene insertion site using site-specific nucleases. This reduces genetic complexity by eliminating redundant or unwanted DNA, while the initial multiple DNA element integration maintains high transformation efficiency during the breeding process.
Solution Approach 2:
The patent applies local quality by using site-specific nucleases with specific recognition sequences to target only the unwanted DNA elements flanking the transgene, while leaving the functional transgene intact. This localized editing reduces genetic complexity without compromising the productivity of the transformation process.
Data Source
AI summary
Transgenic INIR12 maize plants comprising a vip3Aa19 expression cassette linked to a secondary nopaline synthase terminator element which lack a selectable marker gene and/or which comprise modifications that provide for facile excision of the INIR12 transgenic locus from the maize plant genome are provided. Genomic DNA of INIR12 transgenic plants, detection of INIR12 plants and products thereof, methods of making INIR12 plants, and use of INIR12 plants to facilitate breeding are disclosed.