CRISPR Transient Expression Construct for Genome Editing
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Solution Overview
Problem
Current CRISPR/Cas9 and CRISPR/Cpf1 systems for genome editing are labor-intensive due to the need for designing and synthesizing multiple guide-polynucleotides and donor polynucleotides for targeted modifications, especially in multiplex approaches, requiring extensive experimental work and screenings.
Innovation Solution
A CRISPR transient expression construct (CTEC) that expresses a guide-RNA initially and integrates a donor polynucleotide into the target genome, minimizing the risk of guide-RNA integration and reducing the likelihood of gene drives, by using a linear construct with a guide-RNA expression cassette and an additional polynucleotide element that recombines at the target site.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If multiple guide-polynucleotides and donor polynucleotides are designed and synthesized for targeted genome modifications, then the precision of genome editing is improved, but the experimental complexity and labor intensity increase significantly
Solution Approach 1:
The patent combines the guide-polynucleotide and donor polynucleotide into a single integrated construct. The guide-polynucleotide contains both the guide sequence for target recognition and the donor sequence for precise genome modification, eliminating the need to handle separate components and reducing experimental complexity while maintaining editing precision
Solution Approach 2:
The integrated construct serves multiple functions simultaneously: it provides target sequence recognition through the guide portion, enables precise donor polynucleotide incorporation through the donor portion, and facilitates seamless integration into the genome. This multi-functionality reduces the number of separate components needed
2Manufacturing precision
If guide-polynucleotide and donor polynucleotide are used together for targeted modification, then the accuracy of genome editing is improved, but the number of components to be transformed and screened increases
Solution Approach 1:
The guide-polynucleotide and donor polynucleotide are merged into a single integrated construct, reducing the number of separate components from two to one. This maintains the accuracy benefits of having both guide and donor sequences while simplifying transformation and screening procedures
3Stability of the object's composition
If guide-RNA expression cassette is integrated into the genome, then the stability of guide-RNA expression is improved, but the risk of gene drive creation and unintended genomic integration increases
Solution Approach 1:
The patent employs transient expression of the guide-RNA from an episomal or plasmid-based expression cassette rather than permanent genomic integration. The guide-RNA serves its function during the transient expression period and then degrades naturally, avoiding the creation of stable gene drives while maintaining sufficient expression stability for the required duration
Solution Approach 2:
The guide-RNA expression system is designed to be temporary and self-limiting. After fulfilling its genome editing function, the guide-RNA expression cassette is discarded or allowed to degrade naturally, preventing permanent integration and gene drive formation while recovering the benefit of stable expression during the active editing phase
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 simplifies genome editing by reducing the complexity of experimental procedures, minimizing the risk of guide-RNA integration into the genome, and preventing the creation of gene drives, thereby enhancing the efficiency and safety of targeted mutations, insertions, and deletions.
Implementation Method 1
a guide-RNA which is initially expressed from the CTEC and which is capable of binding to a target sequence in a target genome
Implementation Method 2
wherein the CTEC integrates a donor polynucleotide into the target genome at the target sequence, preferably by homologous recombination
Data Source
AI summary
The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention relates to a CRISPR transient expression construct (CTEC) for a genome editing system.