RNA-Templated Plant Genome Editing Beyond Base Editor Limits
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Solution Overview
Problem
Current base editing tools in plants are limited by bystander bases, small editing windows, and inability to edit thymine or guanine residues, restricting their accessibility and versatility in modifying nucleic acids.
Innovation Solution
A method involving a DNA binding domain, DNA endonuclease, and reverse transcriptase, along with CRISPR-Cas nucleases and guide nucleic acids, is used to modify target nucleic acids in plant cells, enabling precise editing and incorporation of desired modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If base editing tools are used to change cytosine and adenine residues, then editing efficiency is improved, but the ability to edit other residues (thymine or guanine) is lost
Solution Approach 1:
The invention divides the editing function into separate modules: a base editor for converting cytosine/adenine to thymine/guanine, and a reverse transcriptase system for incorporating new residues. This segmentation allows each component to specialize in specific editing tasks, thereby expanding the overall editing capability while maintaining efficiency.
Solution Approach 2:
The invention creates a multi-functional editing system where the base editor and reverse transcriptase work together to achieve multiple editing outcomes. The system can convert cytosine to thymine, adenine to guanine, and also incorporate new residues through reverse transcription, making the tool universal for various editing needs.
2Productivity
If base editing tools are used, then cytosine and adenine conversion is achieved, but bystander bases and limited editing windows reduce accessibility
Solution Approach 1:
The reverse transcriptase acts as an intermediary that reads the template strand and synthesizes the edited strand, allowing precise control over the editing process. This intermediary mechanism enables the system to overcome the limitations of direct base editing by using a template-guided approach that can access harder-to-reach targets.
3Adaptability or versatility
If current base editing tools are used, then limited base conversions are achieved, but the ability to convert to residues other than thymine and guanine is lost
Solution Approach 1:
The invention changes the parameter of residue conversion by introducing reverse transcription, which allows the incorporation of any nucleotide residue (A, T, C, or G) at the editing site. This parameter change expands the editing flexibility beyond the limited thymine and guanine conversions of traditional base editors.
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
Enhances the ability to edit nucleic acids in plants beyond cytosine and adenine to thymine and guanine, expanding the range of editable residues and improving editing efficiency.
Implementation Method 1
a reverse transcriptase; and a nucleic acid encoded repair template encoding a modification to be incorporated into the target nucleic acid
Implementation Method 2
a DNA endonuclease (e.g., a first DNA endonuclease)
Implementation Method 3
a CRISPR-Cas nuclease comprising a first DNA binding domain and a first DNA endonuclease
Data Source
AI summary
This invention relates to recombinant nucleic constructs comprising a DNA binding domain, an endonuclease and a reverse transcriptase and methods of use thereof for modifying nucleic acids in plants.