INHT26 Soybean Transgenic Locus Excision via CRISPR
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Solution Overview
Problem
Existing transgenic plants often have undesirable rearrangements and marker genes at transgene insertion sites, which complicates the removal of unnecessary transgenes and integration of new genes, affecting expression and Mendelian transmission of desired traits.
Innovation Solution
The introduction of an originator guide RNA recognition site (OgRRS) in the first DNA junction polynucleotide and a cognate guide RNA recognition site (CgRRS) in the second DNA junction polynucleotide of a DAS44406-6 transgenic locus, allowing for the excision of the INHT26 transgenic locus using an RNA-dependent DNA endonuclease and guide RNA, enabling precise removal of transgenic loci and insertion of new genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If non-site specific integration methods are used to insert transgenes, then the transformation process is simpler and more efficient, but undesirable rearrangements and marker genes remain at insertion sites complicating future modifications
Solution Approach 1:
The patent applies preliminary action by pre-inserting guide RNA recognition sites (gRRS) and protospacer adjacent motifs (PAM sites) at the transgene insertion sites during the initial transformation process. This allows future precise removal or modification of transgenes using CRISPR-Cas systems, converting the complex rearranged insertion sites into controlled, removable structures with predetermined recognition sequences.
Solution Approach 2:
The patent uses guide RNA and Cas proteins as intermediary elements that mediate between the transgene insertion sites and the desired removal or modification outcomes. The gRRS and PAM sites serve as intermediary recognition sequences that enable specific binding and cleavage by CRISPR-Cas systems, facilitating precise control over transgene manipulation without requiring complex site-specific integration methods.
2Reliability
If selectable marker genes are included in transgene insertion sites, then initial selection and identification of transgenic plants is enabled, but removal of these markers becomes difficult and time-consuming
Solution Approach 1:
The patent applies preliminary action by incorporating gRRS and PAM sites flanking the selectable marker genes during initial transgene insertion. This enables future CRISPR-Cas-mediated removal of marker genes through targeted cleavage at predetermined sites, converting the time-consuming manual selection process into a rapid, precise molecular removal approach.
Solution Approach 2:
The patent applies the taking out principle by using CRISPR-Cas systems to extract and remove selectable marker genes from the transgene insertion sites. The gRRS and PAM sites serve as extraction targets, allowing selective removal of marker genes while preserving the functional transgenes, thereby separating the useful traits from the unnecessary markers.
3Reliability
If multiple independent transgene insertions are made to achieve desired traits, then trait expression and Mendelian transmission are improved, but the genetic stability and simplicity of the genome is reduced
Solution Approach 1:
The patent applies merging by consolidating multiple transgene insertions into a single integrated locus where multiple transgenes are co-localized with shared gRRS and PAM sites. This allows simultaneous expression of multiple traits while maintaining a single, stable genomic insertion event, improving Mendelian transmission by ensuring all transgenes are inherited together as a unit.
Solution Approach 2:
The patent applies universality by designing a multi-functional transgene locus where the same gRRS and PAM sites serve multiple purposes: enabling CRISPR-Cas-mediated removal of any transgene, facilitating precise modification of any transgene, and maintaining stable inheritance of all transgenes. This universal design simplifies genome composition while preserving trait expression reliability.
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 allows for the efficient excision of unwanted transgenic loci and insertion of new traits, improving the genetic stability and trait expression in soybean plants, while reducing the presence of selectable marker genes.
Implementation Method 1
excision of the INHT26 transgenic locus using an RNA-dependent DNA endonuclease and guide RNA
Data Source
AI summary
Transgenic INHT26 soybean plants comprising modifications of the DAS44406-6 soybean locus which provide for facile excision of the modified DAS44406-6 transgenic locus or portions thereof, methods of making such plants, and use of such plants to facilitate breeding are disclosed.