Spacer-Specific DNA Recombinases for Precise Genome Integration
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Solution Overview
Problem
Current genome editing methodologies face challenges in achieving precise, site-specific integration of large DNA fragments, requiring inefficient and resource-intensive multi-step processes.
Innovation Solution
A method for generating spacer-specific DNA recombining enzymes (DREs) by evolving amino acid sequences to modify their specificity based on target site spacers, involving expression vector libraries, host cell expression, and selection for active variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional multi-step genome editing processes are used, then site-specific integration can be achieved, but the process becomes time-consuming and resource-intensive
Solution Approach 1:
The patent combines multiple conventional genome editing steps into a single recombination event mediated by spacer-specific recombinases. The enzyme performs integration, excision, and inversion functions that traditionally require separate multi-step processes, thereby improving productivity while maintaining precision through the defined spacer recognition mechanism.
Solution Approach 2:
The invention modifies the specificity parameters of the recombinase enzyme by changing amino acid sequences to recognize custom spacer sequences. This allows the enzyme to be tailored for specific target sites, achieving both high precision integration and improved efficiency by eliminating the need for multiple conventional editing steps.
2Manufacturing precision
If spacer-specific recombinases are engineered, then integration precision at user-defined loci is improved, but the complexity of enzyme generation increases
Solution Approach 1:
The patent uses a library of expression vectors containing variants of the recombinase enzyme, where each vector encodes a version with specific amino acid modifications. By screening this library, the desired spacer-specific recombinase can be isolated and purified, managing the complexity of enzyme generation through systematic variant testing rather than de novo design.
Solution Approach 2:
The invention performs preliminary actions by pre-engineering a library of recombinase variants with different amino acid sequences before actual integration experiments. This allows the specific recombinase needed for a given spacer to be identified and prepared in advance, reducing the overall complexity of the workflow.
3Reliability
If existing recombinase systems are used, then recombination activity is achieved, but adaptability to different spacer sequences is limited
Solution Approach 1:
The patent applies local quality by making specific amino acid modifications in the recombinase enzyme at positions that contact the spacer sequence. These localized changes enable the enzyme to recognize and bind to specific spacer sequences while maintaining the overall recombination function, thereby achieving both reliability and adaptability.
Solution Approach 2:
The invention creates a dynamic system where the recombinase enzyme can be adapted to different spacer sequences through amino acid variant generation. This allows the same basic recombination machinery to be dynamically adjusted for different target sites, enhancing versatility without sacrificing recombination reliability.
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
Enables precise and efficient integration of genetic payloads at user-defined genomic loci, enhancing targeted gene therapy, precise gene therapy, and reducing unwanted effects on cell functions.
Implementation Method 1
Site-specific recombinases (SSRs) are specialized enzymes that promote site-specific DNA rearrangements between defined target regions
Data Source
AI summary
The present invention pertains to a method for generating a spacer-specific DNA recombining enzyme (DRE), the method comprising the steps of: a) providing a library of expression vectors encoding a plurality of variants of a DRE (vDRE), wherein the amino acid sequences of the vDREs comprise one or more amino acid modifications in comparison to a non-variant DRE (nvDRE) from which the vDREs are derived, wherein the nvDRE binds to a first target site comprising in 5′ to 3′ direction a half-site A (HSA), a spacer (Spacer) and a half-site B (HSB), wherein each expression vector comprises: (i) a first region comprising a nucleotide sequence encoding one of the vDREs from among the plurality of vDREs operably linked to an expression control sequence, and (ii) a second region comprising a nucleotide sequence comprising in 5′ to 3′ direction a first target site, an insert nucleotide sequence (INS) of a length at least 1 nucleotide and a second target site, wherein each of the first and second target site comprises the HSA, a variant spacer (vSpacer) and the HSB, wherein the vSpacer differs by at least one nucleotide from the Spacer; b) introducing the library of expression vectors into host cells; c) expressing the plurality of vDREs in the host cells; d) optionally isolating DNA from the host cells; and e) determining whether the vDRE shows activity on the target sites of at least one expression vector.