Ref Nuclease Site-Specific DNA Cleavage via RecA
Solution Overview
Problem
Current methods for cleaving double-stranded DNA at specific locations are limited by the need for engineered recognition sites and often result in non-targeted cleavage due to the availability of restriction enzyme recognition sites, making precise DNA manipulation difficult and time-consuming.
Innovation Solution
The use of N-terminal truncation variants of the Ref protein in combination with RecA and a single-stranded DNA targeting oligonucleotide to create a designer nuclease that can cleave double-stranded DNA at specific sequences without requiring novel zinc finger nucleases or engineered recognition sites, enhancing site-specific targeting activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If restriction enzymes are used to cleave double-stranded DNA at specific locations, then DNA cleavage can be achieved, but cleavage occurs at multiple locations including off-target sites due to limited recognition site specificity
Solution Approach 1:
The invention divides the DNA targeting system into multiple independent components: a single-stranded DNA guide sequence that provides sequence-specific recognition and a nuclease domain that performs cleavage. This segmentation allows the guide sequence to be customized for any target location without being constrained by pre-existing restriction sites, thereby achieving precise single-location cleavage while avoiding off-target effects.
Solution Approach 2:
The invention changes the recognition mechanism from fixed-length palindromic sequences (restriction enzyme recognition sites) to variable-length single-stranded DNA guides that can match any desired target sequence. This parameter change in recognition specificity enables precise targeting at user-defined locations while minimizing unintended cleavage at off-target sites.
2Manufacturing precision
If restriction enzyme recognition sites are engineered into DNA to achieve cleavage at desired locations, then site-specific cleavage can be achieved, but the process becomes difficult and time-consuming
Solution Approach 1:
The invention performs preliminary action by designing the single-stranded DNA guide sequence to directly complement the desired target sequence on the double-stranded DNA. This eliminates the need for preliminary engineering of restriction sites, as the guide sequence can be synthesized directly to match any target location, significantly reducing the time and complexity of site-specific cleavage preparation.
Solution Approach 2:
The invention uses a simplified copying mechanism where the single-stranded DNA guide sequence is designed to be complementary to the target sequence, allowing direct hybridization and cleavage. This copying approach replaces the complex multi-step process of restriction site engineering with a straightforward sequence-complementarity-based system that can be implemented rapidly for any target location.
3Manufacturing precision
If zinc finger nucleases are generated for specific knockdown targets, then precise DNA cleavage can be achieved, but the process becomes difficult and expensive
Solution Approach 1:
The invention segments the zinc finger nuclease system into two independent functional modules: a customizable single-stranded DNA guide sequence that provides sequence-specific recognition and a standardized nuclease domain that performs cleavage. This segmentation eliminates the need to generate novel zinc finger proteins for each target, as the guide sequence can be easily redesigned to target any new location while using the same nuclease component, thereby reducing both complexity and cost.
Solution Approach 2:
The invention creates a universal nuclease system where a single nuclease domain can be paired with different single-stranded DNA guide sequences to target multiple different locations. This multi-functionality allows the same nuclease protein to be reused across numerous different target sites by simply changing the guide sequence, eliminating the need to generate novel nucleases for each target and significantly reducing complexity and expense.
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 allows for precise and efficient cleavage of double-stranded DNA at desired target sequences, reducing off-target cleavage and simplifying the process of introducing specific DNA breaks, thereby facilitating more controlled genetic manipulation.
Implementation Method 1
RecA will bind to the single-stranded DNA targeting fragment to create a nucleoprotein complex. When this complex encounters a homologous double-stranded DNA molecule, the RecA will invade the double-stranded DNA molecule and pair the single-stranded DNA targeting fragment to the complementary sequence in one strand of the double-stranded DNA molecule. The other strand of the duplex (the one identical in sequence to the targeting oligonucleotide) is displaced, and a structure is formed that is often referred to as a D-loop.
Implementation Method 2
In the presence of the RecA-bound and paired DNA targeting fragment, Ref will cleave both strands of the targeted double-stranded DNA molecule at the desired target sequence, within the D-loop.
Data Source
AI summary
Purified Ref polypeptides with increased nuclease site-specific targeting activity, recombinant nucleic acids and cells for expression of such Ref polypeptides, and methods for using the Ref polypeptides in combination with RecA protein and variants thereof to effect targeted nuclease cleavage of a DNA duplex are disclosed.