Recombinogenic Nucleic Acid Strand Vector Design
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Solution Overview
Problem
Current Cas-based techniques for precise modification of nucleic acids are limited by the need to find PAM sites and unique protospacer sequences in the host cell genome, leading to potential off-target cutting and reduced flexibility in targeting desired genomic regions.
Innovation Solution
The method involves using a nucleic acid vector with specifically designed homology arms and intervening sequences, which are cut by Cas nuclease or other cutting means to produce recombinogenic ends, allowing for precise homologous recombination with a donor strand and retrieval or modification of nucleic acid sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If Cas-based techniques are used to precisely modify nucleic acids by finding PAM sites and unique protospacer sequences in the host cell genome, then modification precision is improved, but the flexibility to target desired genomic regions is reduced and off-target cutting risk increases
Solution Approach 1:
The vector is divided into separate functional components: homology arms (HA1 and HA2) that provide targeting specificity, an intervening sequence that contains the retrieval sequence, and cutting sites that are separated from the homology arms. This segmentation allows the homology arms to independently mediate precise targeting while the cutting sites can be optimally positioned for Cas nuclease activity, resolving the contradiction between precision and flexibility.
Solution Approach 2:
The homology arms act as intermediaries between the vector and the donor strand, enabling precise homologous recombination without requiring the Cas nuclease to directly recognize specific genomic sequences. This intermediary mechanism allows flexible targeting of any genomic region with sufficient homology, while the Cas nuclease simply performs cutting at designated sites, separating the precision function from the flexibility function.
2Productivity
If cutting sites are positioned close to homology arms in the vector, then recombination efficiency is improved, but the risk of off-target effects increases due to potential Cas nuclease misalignment
Solution Approach 1:
Different regions of the vector are assigned different functional qualities: the homology arms are designed with high sequence identity to the donor strand for precise pairing, while the cutting sites are positioned in regions with unique sequences that are recognized by the Cas nuclease. This local differentiation ensures that cutting occurs at the correct location without affecting the homology-mediated recombination process, thereby maintaining both efficiency and reliability.
Solution Approach 2:
The vector is pre-designed with cutting sites positioned at optimal locations relative to the homology arms, but the actual cutting is performed only after the vector has been introduced into the cell and the homology arms have annealed to the donor strand. This preliminary positioning followed by delayed cutting ensures that the Cas nuclease acts on the correctly aligned complex, improving reliability while maintaining efficiency.
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 enables precise and flexible targeting of genomic regions, reducing the risk of off-target effects and allowing for the retrieval or modification of large nucleic acid sequences, which is particularly useful for gene therapy and genetic engineering applications.
Implementation Method 1
nuclease cutting of a first cut site (CS1) of the first strand at the 5′ end of HA1, or flanking 5′ of said end; and/or nuclease cutting of a second cut site (CS2) of the first strand at the 3′ end of HA2, or flanking 3′ of said end
Implementation Method 2
carrying out homologous recombination of the cut first strand with the donor strand, whereby gap repair of the first strand produces a vector in which RS is retrieved between HA1 and HA2
Data Source
AI summary
The invention relates to retrieving or modifying target nucleic acids, such as host cell chromosomal DNA, by homologous recombination with vectors that have been cut to provide recombinogenic nucleic acid strands in situ. The methods described herein can be used to modify a target nucleic acid in vitro or in any prokaryotic or eukaryotic cell using homologous recombination.