Exonuclease-Resistant ssDNA Donors for Gene Editing
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Solution Overview
Problem
Current gene editing technologies using engineered site-specific nucleases have low efficiency for targeted gene insertion due to the instability of long single-stranded DNA (ssDNA) and limitations in size, which hampers precise site-specific insertion through the homology-directed repair pathway.
Innovation Solution
The use of ssDNA or dsDNA with exonuclease-resistant modifications, such as biotinylation or phosphorothioate bonds, flanked by homologous arms, and the introduction of site-specific nucleases like Cas9, TALEN, or zinc finger nucleases to create targeted DNA breaks for enhanced gene editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If long ssDNA is used as donor for HDR, then targeted insertion efficiency is improved, but ssDNA stability deteriorates
Solution Approach 1:
The patent applies parameter changes by modifying the chemical structure of ssDNA through exonuclease-resistant modifications (such as phosphorothioate bonds, 2'-O-methyl bases, or 2'-fluoro bases). These modifications change the physical-chemical parameters of the DNA backbone, making it resistant to exonuclease degradation while maintaining its ability to participate in HDR, thus resolving the contradiction between efficiency and stability
Solution Approach 2:
The patent creates a composite structure by combining modified nucleotides with standard nucleotides in the ssDNA donor molecule. The exonuclease-resistant modifications are incorporated at specific positions (such as the 5' end or throughout the sequence) to provide protection against degradation, while the rest of the molecule maintains its natural properties for efficient homologous recombination
2Productivity
If ssDNA size is increased for better HDR, then targeted insertion efficiency is improved, but synthesis feasibility deteriorates
Solution Approach 1:
The patent applies segmentation by dividing the long ssDNA donor into smaller oligonucleotide fragments that can be chemically synthesized and then annealed together. This allows the final donor molecule to have a large effective size for high HDR efficiency while each individual synthesized fragment remains within the feasible size limit for chemical synthesis (typically <200 nucleotides)
Solution Approach 2:
The patent employs preliminary action by first synthesizing smaller, exonuclease-resistant oligonucleotide fragments with modified bases, then annealing them to form the complete long ssDNA donor. This preliminary synthesis of protected fragments ensures that the final assembled molecule has both the required length for efficient HDR and the stability conferred by the modifications
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 increases the efficiency of targeted gene insertion by stabilizing ssDNA and dsDNA with 3' single-stranded overhangs, allowing for more precise and efficient repair mechanisms, thereby improving the overall gene editing process.
Implementation Method 1
the ssDNA has an exonuclease resistant modification
Implementation Method 2
the nuclease to create a DNA double-strand break (DSB)
Implementation Method 3
Precise site-specific insertion of a gene of interest usually happens through the homology directed repair (HDR) pathway
Data Source
AI summary
The present disclosure provides compositions and methods for targeted insertion of a gene of interest in the genome of a cell using single-stranded DNA or double-stranded DNA with 3 overhang. Also provided are methods of generating single-stranded DNA or double-stranded DNA with 3′ over-hang that can be used for targeted insertion.