Haploid Inducer Pollen Delivery for Germline Gene Editing
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Solution Overview
Problem
Existing methods for gene editing in recalcitrant crop varieties are laborious, time-consuming, and introduce genetic impurities due to linkage, while transient methods lack efficiency in ensuring germ-line edits are passed to progeny.
Innovation Solution
Utilizing haploid inducing lines to transiently introduce gene editing machinery during haploid induction, allowing simultaneous editing and haploid induction in crops, eliminating the need for tissue culture and ensuring edits are passed to progeny.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If transformation methods (Agrobacterium or biolistic) are used to introduce editing machinery into plants, then gene editing can be achieved, but the process becomes genotype-dependent and cannot work for all crops or varieties
Solution Approach 1:
The patent uses pollen as an intermediary vehicle to deliver editing machinery (Cas9 and guide RNA) from the haploid inducer parent to the target plant. This natural biological mediator bypasses the need for direct transformation of recalcitrant varieties, enabling gene editing in crops that are otherwise difficult or impossible to transform using conventional methods.
Solution Approach 2:
The haploid inducer line serves multiple functions simultaneously: it induces haploid development in the target plant and delivers the gene editing machinery through its pollen. This multi-functional approach allows a single system to overcome both the transformation recalcitrance and enable gene editing across diverse crop varieties without requiring variety-specific optimization.
2Manufacturing precision
If trait introgression is used to introduce edits in recalcitrant varieties, then gene editing can be achieved, but the process is expensive, laborious, time-consuming, and introduces genetic impurity due to linkage
Solution Approach 1:
The patent extracts only the essential editing machinery (Cas9 and guide RNA) from the haploid inducer parent through pollen delivery, while the rest of the inducer genome is eliminated during haploid development. This selective transfer achieves precise gene editing without introducing unwanted linked genes, eliminating the genetic impurity problem associated with traditional trait introgression.
Solution Approach 2:
The patent segments the genetic material transfer process by separating the editing machinery delivery (through pollen) from the rest of the genome (which is eliminated during haploid induction). This segmentation allows precise introduction of only the desired editing components without the cumbersome linked blocks that characterize traditional introgression methods.
3Ease of operation
If floral dipping or transient transformation is used to introduce editing machinery without tissue culture, then the method is simpler, but there is no established routine method to ensure edits end up in germ-line cells
Solution Approach 1:
The patent performs preliminary action by delivering the editing machinery through pollen during the natural fertilization process, ensuring the Cas9 and guide RNA are present in the zygote and early embryonic cells before germ-line specification occurs. This timing ensures high probability of germ-line editing without requiring complex post-transformation selection or tissue culture procedures.
Solution Approach 2:
The haploid induction process itself serves the dual function of delivering editing machinery and ensuring germ-line incorporation. The natural haploid induction mechanism automatically selects for plants that have received and incorporated the editing components, eliminating the need for external selection markers or complex screening procedures required in conventional transformation methods.
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 rapid, cost-effective gene editing in elite lines without tissue culture, producing edited doubled haploid lines suitable for breeding programs.
Implementation Method 1
using a haploid inducing line (whether existing or created) and transforming the haploid inducing line so that it contains DNA coding for cellular machinery capable of editing genes
Implementation Method 2
DNA coding for the editing machinery (e.g., CAS9 and guide RNA) is introduced into plant callus, seed or embryonic tissue
Implementation Method 3
Haploid induction ('HI') is a class of plant phenomena characterized by loss of one parent's set of chromosomes (the chromosomes from the haploid inducer parent) from the embryo at some time during or after fertilization
Implementation Method 4
One way to do this is to provide a donor template that has the desired mutation and sequence homology to the region surrounding the target site
Data Source
AI summary
The presently disclosed subject matter relates to using a haploid inducing line (whether existing or created) and transforming the haploid line so that it encodes cellular machinery capable of editing genes. The transformed haploid inducing line is used as a parent in a cross between two plants. During pollination, the parental gametes fuse to form an embryo; and the gene editing machinery is also delivered to the embryo at this time. During embryonic development, one set of parental chromosomes are lost, and the gene editing machinery operates on the remaining set of chromosomes. Thus, at least one haploid progeny with edited genes is produced from the cross.