Multiplex Guide Polynucleotide Expression Using RNase III Cleavage
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Solution Overview
Problem
Existing CRISPR-based genome editing methods struggle to efficiently deliver and process multiple guide polynucleotides for multiplexed editing of polygenic plant traits, which require coordinated effects of numerous genes.
Innovation Solution
A method and composition that utilize eukaryotic RNase III, heterologous RNase III, or double-stranded RNA-specific proteins to cleave RNA molecules containing stem loops, enabling the simultaneous delivery and expression of multiple guide polynucleotides from a single transcript in plant cells, using Cas endonucleases for targeted modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple guide polynucleotides are delivered separately, then each guide can be optimized independently, but the delivery complexity and time required increase significantly
Solution Approach 1:
The patent combines multiple guide polynucleotide sequences into a single polynucleotide molecule with a shared promoter and regulatory elements. This merging approach allows simultaneous delivery of multiple guides through a single transformation event, reducing delivery complexity while maintaining the ability to target multiple genomic locations for CRISPR editing.
Solution Approach 2:
The polynucleotide molecule is segmented into distinct functional regions: promoter sequences, individual guide polynucleotide sequences separated by spacer regions, and termination signals. This segmentation allows each guide to be independently designed and optimized while being delivered as an integrated unit, resolving the contradiction between independent optimization and delivery simplicity.
2Ease of operation
If a single polynucleotide molecule contains multiple guide sequences, then delivery is simplified, but the RNA processing complexity increases
Solution Approach 1:
The patent introduces eukaryotic RNase III as an intermediary enzyme that processes the polynucleotide molecule by cleaving it at specific recognition sequences. This mediator enzyme simplifies the overall system by providing a controlled, predictable mechanism for generating individual guide RNAs from the multi-guide polynucleotide, reducing the complexity of RNA processing while maintaining delivery simplicity.
Solution Approach 2:
The patent utilizes changes in RNA secondary structure parameters, specifically stem-loop formations at RNase III recognition sites, to control processing specificity. By designing the RNA molecule with specific structural features (stem loops) at cleavage sites, the system achieves precise control over where processing occurs, simplifying the processing complexity while maintaining ease of operation.
3Reliability
If eukaryotic RNase III is used to cleave the RNA molecule, then guide polynucleotide processing is achieved, but heterologous expression requirements increase
Solution Approach 1:
The patent employs eukaryotic RNase III, which is universally present in eukaryotic cells including plants, animals, and fungi. This universal enzyme can process the guide polynucleotide in multiple host species without requiring host-specific enzymes, thereby achieving reliable processing while maintaining broad host cell compatibility and adaptability across different eukaryotic systems.
Solution Approach 2:
The patent uses a heterologous RNase III enzyme that can be expressed in the host cell to perform the cleavage function. By using an enzyme from a different species (heterologous expression), the system achieves reliable processing while the enzyme can be produced in various host cells, maintaining versatility. The RNase III gene can be cloned and expressed in different eukaryotic hosts to generate the necessary cleavage activity.
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
Facilitates efficient multiplexed genome editing in plants by delivering and processing multiple guide polynucleotides, allowing for coordinated genetic modifications across multiple genomic targets, enhancing the precision and efficiency of trait improvement.
Implementation Method 1
expressing a eukaryotic RNase III in the plant cell to cleave an RNA molecule transcribed from the polynucleotide molecule
Implementation Method 2
the RNA molecule having the RNase III recognition sequence comprises at least one stem loop
Data Source
AI summary
The present disclosure relates to methods and compositions for expressing multiple guide polynucleotides from one or more transcripts. Compositions and methods for delivering a plurality of guide polynucleotides to target multiple independent sites in a cell's genome are also provided.