Linear Non-Homologous DNA for Enhanced Genome Editing Indels
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Solution Overview
Problem
Current methods for targeted genome editing, such as using zinc finger nucleases, TALENs, and CRISPR/Cas proteins, face limitations in the frequency of generating desired mutations like indels due to the efficiency of genome cutting and repair events, which hampers the process of disrupting specific genes in eukaryotic cells.
Innovation Solution
Incorporating a linearized non-homologous DNA composition with a genome targeting composition that includes a genome editing endonuclease, such as a zinc finger nuclease or a CRISPR/Cas endonuclease, to increase the frequency of insertions and deletions (indels) by promoting error-prone end-joining repair mechanisms in eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If genome editing endonuclease is used to induce insertion and deletion mutations (indels), then gene disruption is achieved, but error-free repair occurs in some cells reducing the frequency of desired indels
Solution Approach 1:
The patent introduces a linear non-homologous DNA composition as an intermediary substance that competes with the genomic DNA for error-free NHEJ repair. This mediator DNA absorbs the error-free repair capacity of the cell, thereby increasing the frequency of indels in the target genomic sequence while maintaining the natural genome editing endonuclease mechanism.
2Measurement precision
If clones are screened to find desired mutations, then gene disruption can be identified, but the process is limited by the number of clones screened and the frequency of repair events
Solution Approach 1:
The patent changes the parameter of repair outcome frequency by introducing the linear non-homologous DNA composition. This shifts the repair outcome distribution toward error-prone NHEJ, increasing indel frequency from typical low levels to significantly higher levels, thereby reducing the number of clones that need to be screened and the time required to identify desired mutations.
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 significantly enhances the rate of indel generation in targeted genomic DNA, increasing the likelihood of successful gene editing by reducing error-free non-homologous end-joining and thereby increasing the number of cells with desired mutations.
Implementation Method 1
a genome editing endonuclease (e.g., a zinc finger nuclease, a TALEN, a class 2 CRISPR/Cas endonuclease, etc.) to induce insertion and deletion mutations (indels) in the genomic DNA
Implementation Method 2
the cell's DNA repair machinery repairs the lesion either by non-homologous end joining (NHEJ) or homology-directed recombination (HDR)
Implementation Method 3
the cell's DNA repair machinery repairs the lesion either by non-homologous end joining (NHEJ) or homology-directed recombination (HDR)
Implementation Method 4
including a linear non-homologous DNA composition increases the likelihood of error-prone end-joining. In other words, the presence of a linear non-homologous DNA composition decreases the rate of error-free NHEJ, and thereby increases the rate of insertions and deletions (indels) that are produced in the target DNA
Data Source
AI summary
The present disclosure provides methods and compositions for enhanced editing of genomic DNA. For example, in some embodiments, a subject method is a method of editing genomic DNA of a eukaryotic cell and the method includes introducing into a eukaryotic cell a composition comprising: (a) a linearized non-homologous DNA composition and (b) a genome targeting composition (which includes a genome editing endonuclease, or a nucleic acid encoding the genome editing endonuclease). In some cases in which the genome editing endonuclease is a CRISPR/Cas endonuclease, the genome targeting composition can also include a corresponding CRISPR/Cas guide RNA.