Recombinase Allele Constructs for Single-Step Conditional Knockouts
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Solution Overview
Problem
Existing methods for generating genetically modified organisms with multifunctional loci, such as true KO-first and conditional-null alleles, have had mixed success and often fail to achieve desired functionalities.
Innovation Solution
A nucleic acid construct design using specific recombinase recognition sites and sequences allows for the simultaneous deletion and inversion of genetic elements, enabling the creation of multifunctional alleles in a single targeting step, including a 3′ splice region, actuating sequence, nucleotide sequence of interest, and a COIN, with controlled orientations and recombinase treatments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional knockout methods with selection cassettes are used, then gene deletion can be achieved, but the process requires multiple steps including selection cassette insertion and subsequent removal
Solution Approach 1:
The invention extracts and removes the selection cassette from the final modified allele, keeping only the essential elements (reporter and floxed exon). This allows the knockout function to be achieved without the complexity of selection cassette management in the final product.
Solution Approach 2:
The selection cassette is pre-inserted into the targeting vector to enable efficient selection of correctly targeted cells during the gene targeting process. After selection, the cassette is removed, having served its purpose only during the targeting phase.
2Adaptability or versatility
If conditional alleles are created using floxed exons, then conditional gene control is achieved, but the presence of residual loxP sites and reporters may interfere with downstream applications
Solution Approach 1:
The invention uses the reporter element's presence as a beneficial feature for detecting allele status and confirming proper gene targeting, rather than treating it as a harmful residual element. The reporter provides useful information about the modified allele's state.
3Productivity
If multiple functionalities (knockout and conditional) are incorporated in one targeting vector, then the number of targeting steps is reduced, but the functionality and reliability of the resulting alleles are compromised
Solution Approach 1:
The invention segments the modified allele into distinct functional modules: a reporter module, a floxed exon module, and a selection cassette module. This segmentation allows each element to perform its specific function independently and reliably, even when present in the same allele.
4Reliability
If selection cassettes are permanently retained in modified alleles, then selection pressure can be maintained, but the genetic modification becomes more complex and less versatile
Solution Approach 1:
The invention makes the selection cassette dynamic - present and active during the targeting phase when selection is needed, then removable afterward when it is no longer needed. This dynamic presence/absence allows the system to adapt to different phases of the gene modification process.
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 the efficient generation of genetically modified organisms with true KO-first and conditional-null alleles, ensuring functional reporter expression and conditional control in a single targeting step, thereby improving the reliability and effectiveness of genetic modification.
Implementation Method 1
A nucleic acid construct design using specific recombinase recognition sites and sequences allows for the simultaneous deletion and inversion of genetic elements
Data Source
AI summary
Methods for modifying a genome are provided, wherein the modifications comprise null alleles, conditional alleles and null alleles comprising conditional by inversion elements. Methods are provided which afford the ability in a single targeting step to introduce an allele that can be used to generate a null allele, a conditional allele, or an allele that is a null allele and that further includes a conditional by inversion element. Introduced alleles comprise pairs of cognate recombinase recognition sites, an actuating sequence and/or a drug selection cassette, and a nucleotide sequence of interest, and a conditional by inversion element, wherein upon action of a recombinase a conditional allele with a conditional by inversion element is formed. In a further embodiment, action of a second recombinase forms an allele that contains only a conditional by inversion element in sense orientation. In a further embodiment, action by a third recombinase forms an allele that contains only the actuating sequence in sense orientation.