Bacillus Genome Integration via Linear DNA and Cas9
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Solution Overview
Problem
Current methods for integrating donor DNA sequences into the genome of Bacillus sp. cells require the use of selectable markers and rely on spontaneous DNA damage, which are inefficient and limited by rare events, and lack flexibility in targeting specific genomic locations.
Innovation Solution
A method involving a linear recombinant DNA construct with donor DNA sequences flanked by long homology arms (>1000 nucleotides) combined with a circular construct encoding a Cas9 endonuclease and guide RNA, which introduces site-specific DNA damage, allowing for efficient integration without the need for selectable markers and increased targeting precision.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If spontaneous double strand break occurrence is used for gene integration, then integration can occur without selectable markers, but the integration frequency is extremely low and relies on rare events
Solution Approach 1:
The patent applies preliminary action by pre-introducing the donor DNA fragment with homology arms into the bacterial cell before the spontaneous double strand break occurs. The donor DNA is prepared in advance with specific homology sequences that will facilitate subsequent integration, transforming a passive rare event into an active controlled process that significantly increases integration frequency without requiring selectable markers.
2Reliability
If selectable markers are integrated into the genome to identify transformed cells, then transformation efficiency can be selected for, but the flexibility in targeting specific genomic locations is reduced
Solution Approach 1:
The patent segments the DNA construct into distinct functional components: the donor DNA fragment containing the gene of interest flanked by homology arms, and the Cas9-gRNA complex for targeted cleavage. This segmentation allows the homology arms to provide targeting flexibility while the donor DNA provides the functional gene, eliminating the need for selectable markers to be integrated into the genome and thus maintaining both reliability and adaptability.
3Device complexity
If short homology arms are used in linear DNA fragments, then the DNA structure is simpler, but the integration relies on rare spontaneous DNA damage and cannot achieve site-specific integration
Solution Approach 1:
The patent changes the parameter of homology arm length from short to long (greater than 1000 nucleotides). This parameter change fundamentally alters the integration mechanism from relying on rare spontaneous breaks to enabling homology-directed repair, achieving site-specific integration precision while maintaining reasonable construct complexity through the use of long homology arms that facilitate precise recombination.
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 significantly enhances the frequency of donor DNA integration into the Bacillus sp. genome, offering higher efficiency and flexibility in targeting specific genomic locations without integrating selectable markers, thus overcoming the limitations of previous methods.
Implementation Method 1
combining the crRNA with a Cas endonuclease (through any convenient and conventional means) into a functional complex in a host cell. The sequence of the RNA component of Cas9 can be designed such that Cas9 recognizes and cleaves DNA containing (i) sequence complementary to a portion of the RNA component and (ii) a protospacer adjacent motif (PAM) sequence.
Implementation Method 2
The present disclosure includes methods and compositions for integrating donor DNA sequences into a target site on the genome of a Bacillus sp. cell without the integration of a selectable marker into said genome. The methods employ a linear recombinant DNA construct comprising a donor DNA sequence flanked by long homology arms (greater than 1000 nucleotides in length)
Data Source
AI summary
Methods and compositions are provided for integrating donor DNA sequences into the genome of a Bacillus sp. cell without the integration of a selectable marker into said genome. The methods employ a linear recombinant DNA construct comprising a donor DNA flanked by long homology arms (each of at least 1000 nucleotides in length) in combination with a recombinant DNA construct encoding a Cas9 endonuclease and a guide RNA, for the introduction of a guide RNA/Cas endonuclease into a Bacillus sp. cell, and as such providing a highly effective system for integrating donor DNA sequences into the genome of said Bacillus sp. cell, without the need to integrate a selectable marker in the genome of said Bacillus sp. cell.