CRISPR/Cas9 Genome Editing via Single-Stranded Oligo Donors
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Solution Overview
Problem
Conventional methods for producing genome-edited mice, such as conditional knock-out mice, are inefficient and time-consuming, particularly when attempting to introduce loxP sequences at multiple sites, often requiring skilled techniques and taking several months to years to complete.
Innovation Solution
An in vitro genome editing method using a CRISPR/Cas system with an artificial nuclease system and single-stranded DNA containing 5'-side homology arm sequences, donor DNA sequences, and 3'-side homology arm sequences, introduced via electroporation, enables efficient genome editing by cleaving both ends of the genome editing target region and facilitating the introduction of donor DNA sequences at multiple sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional methods (ES cells and homologous recombination) are used to produce conditional knock-out mice, then the production process is well-established, but it takes one to two years to finally produce conditional mice
Solution Approach 1:
The patent changes the fundamental parameters of the genome editing approach by transitioning from homologous recombination in ES cells to CRISPR/Cas9-mediated direct editing in fertilized eggs. This parameter change includes using RNA-guided nucleases instead of homologous recombination, injecting CRISPR components (Cas9 protein/mRNA and guide RNA) directly into zygotes, and utilizing single-stranded oligo donors for efficient knock-in, thereby reducing production time from 1-2 years to several months while maintaining reliability
Solution Approach 2:
The patent performs preliminary actions by preparing and injecting all necessary CRISPR components (Cas9 protein, mRNA, guide RNA, and single-stranded oligo donor sequences) into fertilized eggs before embryonic development begins. This preliminary setup of the editing machinery and donor templates in the zygote stage enables direct genome modification without requiring subsequent ES cell manipulation, chimeric mouse generation, or breeding steps, thus dramatically accelerating the production timeline
2Productivity
If CRISPR/Cas system is used to knock in loxP sequences at two sites simultaneously, then production efficiency should improve, but in practice it often happens that a loxP sequence is knocked in only at one site
Solution Approach 1:
The patent introduces single-stranded oligo DNA sequences as intermediary donor templates that contain the loxP sequences and flanking homology regions. These oligos serve as mediators that facilitate precise dual-site knock-in by providing the necessary DNA template for homologous recombination at both target sites simultaneously. The use of these intermediary oligos, combined with optimized CRISPR guide RNA design and delivery conditions, ensures high-fidelity dual-site editing while maintaining high productivity
Solution Approach 2:
The patent employs partial or excessive action by using multiple CRISPR guide RNAs targeting both sites, along with single-stranded oligo donors that contain extended homology regions beyond the minimal required sequences. This excessive provision of editing components and donor templates ensures that both sites are successfully edited simultaneously, overcoming the limitation of single-site knock-in that occurs with conventional approaches
3Manufacturing precision
If conventional donor vectors are used to insert loxP sequences at two sites, then it is theoretically possible to achieve dual knock-in, but the efficiency with which flox mice are obtained is not high
Solution Approach 1:
The patent replaces complex, difficult-to-deliver conventional donor vectors with simple, easy-to-deliver single-stranded oligo DNA sequences. These oligos are synthesized in vitro, are stable enough for injection, but do not require complex vector construction or viral transduction. They serve as disposable donor templates that are efficiently taken up by the zygote and used for precise knock-in at both sites, dramatically improving flox mouse production efficiency while maintaining dual-site insertion capability
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 significantly reduces the production time of genome-edited mice to three to six months, lowers production costs, and enhances the efficiency of introducing mutations in relatively long regions or multiple sites, making it suitable for large-scale projects like the production of conditional knock-out mice.
Implementation Method 1
When Cas mRNA and guide RNA are injected into a fertilized egg, the guide RNA binds to the target site, and the Cas protein induced at the binding site performs double-strand cleavage of DNA
Implementation Method 2
introduced via electroporation
Data Source
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AI summary
A method for producing a genome edited cell or a non-human organism, comprising the step of introducing (a) an artificial nuclease system which cleaves both ends of a genome editing target region, and (b) a nucleic acid sequence formed by arranging a 5'-side homology arm sequence, a donor DNA sequence, and a 3'-side homology arm sequence in this order from a 5'-side, the 5'-side homology arm sequence being a homologous sequence of one nucleic acid sequence outside the genome editing target region, and the 3'-side homology arm sequence being a homologous sequence of the other nucleic acid sequence outside the genome editing target region, into a cell or a non-human organism.