Single-Stranded DNA Stabilization for Genome Editing
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Solution Overview
Problem
Current methods for genome editing, particularly using CRISPR and TALENs, face inefficiencies in both NHEJ and HDR pathways due to challenges in synthesizing large ssDNA and stabilizing ssDNA during digestion, leading to low editing precision and high error rates.
Innovation Solution
Incorporating a non-specific DNA binding protein, such as single-stranded binding protein (SSB), into genome editing reactions to enhance the efficiency of both NHEJ and HDR-mediated editing, and using a combination of exonucleases like lambda and exonuclease III to efficiently produce ssDNA from dsDNA without buffer changes or heat inactivation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If large ssDNA is synthesized for HDR, then homology-directed repair efficiency should improve, but synthesis difficulty and cost increase significantly
Solution Approach 1:
The patent segments the ssDNA synthesis process into two distinct phases: first synthesizing dsDNA template (easy to manufacture), then converting to ssDNA in situ within cells using RecA-mediated strand invasion. This avoids the difficulty of directly synthesizing large ssDNA while achieving the same functional result for HDR.
Solution Approach 2:
The patent introduces RecA protein as an intermediary that facilitates the conversion from dsDNA to functional ssDNA for HDR. RecA mediates strand invasion and homologous pairing, allowing the cell to generate the required ssDNA structure from dsDNA template without external synthesis intervention.
2Productivity
If exonucleases are used to generate ssDNA from dsDNA, then ssDNA availability for HDR improves, but exonuclease stalling causes partial digestion and reduces efficiency
Solution Approach 1:
The patent extracts the problematic exonuclease digestion step from the overall ssDNA generation process and replaces it with RecA-mediated strand invasion. This removes the source of stalling and partial digestion while achieving the same goal of providing ssDNA for HDR through an alternative mechanism.
Solution Approach 2:
The patent enables the cell's own RecA protein to perform the ssDNA generation function that was previously attempted using external exonucleases. The cellular machinery itself services the need for ssDNA by mediating strand invasion from the introduced dsDNA template, ensuring complete and reliable conversion.
3Speed
If NHEJ pathway is used for genome editing, then editing speed is fast, but precision and accuracy deteriorate due to error-prone nature
Solution Approach 1:
The patent dynamically shifts the cellular repair pathway preference from NHEJ to HDR by providing exogenous dsDNA templates with homology arms and utilizing RecA overexpression. This dynamic intervention allows the cell to switch from the fast but error-prone NHEJ pathway to the slower but precise HDR pathway, achieving both speed control and precision improvement.
Solution Approach 2:
The patent changes key parameters including providing dsDNA templates with extended homology arms (changing template structure), overexpressing RecA (changing protein concentration), and optimizing delivery methods (changing delivery parameters). These parameter changes collectively shift the repair pathway balance toward HDR, improving precision while maintaining acceptable editing speeds.
4Manufacturing precision
If HDR pathway is used for precise genome editing, then editing precision improves, but editing efficiency remains low compared to NHEJ
Solution Approach 1:
The patent performs preliminary actions by pre-synthesizing dsDNA templates with optimized homology arms and pre-expressing RecA protein before inducing the DSB. This preliminary preparation ensures that when HDR is activated, the cellular machinery is already primed and ready, significantly improving HDR efficiency without sacrificing precision.
Solution Approach 2:
The patent uses a composite approach combining dsDNA template (with homology arms), RecA protein, and CRISPR-Cas9 system together. This composite system works synergistically: the dsDNA provides the repair template, RecA facilitates homologous pairing and strand invasion, and CRISPR-Cas9 creates the DSB, collectively achieving both high precision and high efficiency in HDR-mediated editing.
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
The approach significantly improves genome editing efficiency by stabilizing ssDNA and reducing errors, allowing for precise and efficient integration of donor DNA during HDR, while minimizing off-target effects and degradation.
Implementation Method 1
contacting the composition with a first exonuclease (e.g., a 5′ to 3′ exonuclease such as lambda exonuclease, or the like); and (c) contacting the composition comprising the first exonuclease with a second exonuclease (e.g., a 3′ to 5′ exonuclease such as exonuclease III, or the like or a 5′ to 3′ exonuclease such as T7 exonuclease, or the like)
Implementation Method 2
addition of a non-specific DNA binding protein (e.g., a DNA binding protein that does not target a specific sequence, such as single stranded binding protein) to a genome editing reaction increased the efficiency of both NHEJ-mediated and HDR-mediated genome editing
Data Source
AI summary
The present disclosure relates, in part, to improved methods of making single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA), as well as use of the resulting ssDNA for genome engineering. The disclosure also relates, in part, to improved methods of genetic modification using single stranded DNA binding proteins.