Extended Guide RNA Editing for Broader Plant Allele Replacement
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Solution Overview
Problem
Current base editing tools are limited in their ability to edit nucleic acids beyond changing cytosine and adenine to thymine and guanine, have restricted accessibility due to high PAM density requirements, and cannot edit thymine or guanine residues, limiting their applicability to trait-relevant targets.
Innovation Solution
The use of Type V CRISPR-Cas effector proteins and reverse transcriptases, combined with extended guide nucleic acids, to modify target nucleic acids by introducing extended guide nucleic acids and employing a first and second CRISPR-Cas effector proteins with a reverse transcriptase to facilitate broader editing capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If current base editing tools are used, then cytosine and adenine residues can be converted to thymine and guanine, but the ability to edit other residues is limited
Solution Approach 1:
The guide RNA is segmented into distinct functional domains: a spacer region for target recognition and an extended region containing the edit template. This segmentation allows the system to separately optimize for target specificity and editing versatility, enabling conversion of multiple residue types while maintaining accuracy through the modular architecture
Solution Approach 2:
An extended guide RNA acts as an intermediary between the Cas12a effector protein and the target DNA. This intermediary contains both the target-binding spacer and the edit template, mediating the transfer of editing information while enabling versatile residue conversion through the incorporated template sequence
2Manufacturing precision
If base editing is performed with current tools, then specific base conversions are achieved, but bystander bases are affected
Solution Approach 1:
The editing system applies local quality by restricting modifications to only the specific position indicated in the edit template within the extended guide RNA. The Cas12a-nickase introduces a nick at a defined location, and the extended guide RNA provides a template that directs insertion or deletion at this precise site, ensuring that only the intended base is modified while bystander bases remain unaffected
Solution Approach 2:
Instead of using a Cas9 endonuclease that creates a double-strand break affecting multiple bases, the system uses Cas12a-nickase that creates a single-strand nick. This inverted approach (single nick instead of double-strand break) combined with the extended guide RNA template enables precise editing at the nick site without affecting adjacent bases, thereby eliminating bystander base effects
3Manufacturing precision
If base editing windows are made smaller for precision, then editing accuracy improves, but accessibility to trait-relevant targets is limited
Solution Approach 1:
The extended guide RNA structure provides multi-functionality by combining target recognition (spacer region) with editing template provision (extended region). This universal design allows the same Cas12a-nickase complex to achieve both precise targeting and versatile editing outcomes, making the system adaptable to a broad range of trait-relevant targets while maintaining high precision through the defined nick site and template-directed repair
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 approach enhances the range of possible edits in nucleic acids, allowing for more versatile and efficient modification of nucleic acids in various organisms, including plants, by increasing the accessibility of trait-relevant targets.
Implementation Method 1
contacting the target nucleic acid with (a) a Type V CRISPR-Cas effector protein or a Type II CRISPR-Cas effector protein; (b) a reverse transcriptase, and (c) an extended guide nucleic acid
Data Source
AI summary
This invention relates to recombinant nucleic constructs comprising Type II CRISPR-Cas effector proteins, reverse transcriptases and extended guide nucleic acids and methods of use thereof for modifying nucleic acids in plants.