Linear Donor DNA for CRISPR Gene Knockout Enrichment
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Solution Overview
Problem
Current genome editing technologies, particularly the CRISPR/Cas9 system, face challenges in efficiently generating gene knockouts in mammalian cells, especially for multi-gene knockouts, due to low enrichment of rare clones with target gene modifications and the complexity of designing effective guide RNAs for certain genes.
Innovation Solution
A donor construct comprising a linear double-stranded DNA with a marker gene is introduced into cells, featuring protective sequences and target sites cleavable by sequence-specific nucleases like Cas9, allowing for efficient insertion by non-homologous end joining and enrichment of gene-knockout cells through marker selection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional CRISPR/Cas9 system is used for gene knockout, then sequence-specificity is achieved, but enrichment efficiency of rare clones with target gene modifications is low
Solution Approach 1:
The patent introduces a donor DNA construct as an intermediary element that mediates between the CRISPR/Cas9 system and the target gene. The donor DNA contains a selectable marker gene flanked by homology arms that match the target locus, enabling homologous recombination. This intermediary facilitates efficient enrichment of edited clones through marker selection while maintaining the sequence-specificity of CRISPR/Cas9-guided cleavage.
Solution Approach 2:
The patent employs preliminary action by pre-designing and introducing the donor DNA construct containing the selectable marker before selecting edited clones. The marker gene is预先 integrated into the donor construct with homology arms, so that when homologous recombination occurs at the target site, the marker is automatically incorporated. This preliminary preparation enables direct enrichment of edited cells through marker selection, dramatically improving productivity.
2Adaptability or versatility
If CRISPR/Cas9 system is used for multi-gene knockout, then genome editing capability is achieved, but the task becomes time-consuming and complex
Solution Approach 1:
The patent applies segmentation by dividing the multi-gene knockout task into multiple independent donor DNA constructs, each targeting a specific gene. Each construct contains a unique selectable marker and homology arms specific to its target gene. This segmentation allows parallel design and introduction of multiple gene-targeting systems, reducing the time and complexity compared to sequential editing approaches.
Solution Approach 2:
The patent creates a universal platform for multi-gene knockout by using a standardized donor DNA construct design. The same basic structure (selectable marker + homology arms) can be applied to any target gene by simply changing the homology arm sequences. This multi-functional approach enables the same methodology to be used for single or multiple gene knockouts, significantly improving efficiency and reducing time investment.
3Manufacturing precision
If guide RNA is designed for certain genes, then sequence-specific cleavage is achieved, but design becomes difficult for some genes
Solution Approach 1:
The patent uses the donor DNA construct as an intermediary that bypasses the need for optimal guide RNA design in some cases. The donor DNA contains pre-designed homology arms that directly match the target gene sequences, allowing homologous recombination to occur even when guide RNA design is challenging. This intermediary approach maintains sequence-specificity while reducing design difficulty.
4Device complexity
If traditional methods are used for gene knockout, then simplicity is maintained, but enrichment for rare clones is ineffective
Solution Approach 1:
The patent introduces a donor DNA construct as an intermediary that carries a selectable marker gene. This simple addition to the traditional CRISPR approach enables effective enrichment of rare edited clones through marker-based selection, dramatically improving productivity while maintaining the overall simplicity of the methodology.
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 enhances the efficiency of gene knockout by enabling the enrichment of cells with specific modifications, improving the generation of both single and multi-gene knockouts, even in cases where guide RNA design is difficult, and expands the application of genome editing systems like CRISPR in biomedicine.
Implementation Method 1
a sequence-specific nuclease that can cleave a target site in a cell genome is used to also cleave at least one target site contained in the linear donor DNA
Implementation Method 2
The linear donor DNA is inserted into the cleaved target site in the cell genome through a non-homologous end joining repair mechanism
Data Source
AI summary
A donor construct and a gene knockout method, as well as a system and kit for the gene knockout are provided. The donor construct is a linear donor DNA or can be cleaved in a cell to produce the linear donor DNA. The gene knockout method uses a marker gene contained in the donor construct to enrich cells in which a gene is knocked out, thereby improving the efficiency of generating the gene knockout by a sequence-specific nuclease.