CRISPR-Cas9 Gene Drive for Rodent Genotype Conversion
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Solution Overview
Problem
Current methods for producing desired mutant genotypes in rodents are time-consuming and costly, especially when multiple loci are involved, and are exacerbated by genetic linkage, requiring numerous animals and generations.
Innovation Solution
The implementation of a CRISPR-Cas9 mediated gene drive system that leverages homology-directed repair to convert heterozygous genotypes to homozygosity in a single generation, utilizing a split gene-drive system with separate elements for Cas9 and guide RNAs to efficiently introduce and transmit desired alleles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If Mendelian genetics breeding is used to combine multiple loci mutations, then desired compound mutant genotypes can be produced, but the process requires substantial number of animals, time, and money
Solution Approach 1:
The gene drive system enables self-propagation of desired alleles through the population. The CRISPR-Cas9 mechanism automatically copies the drive allele from one chromosome to its homologous counterpart in the germline, eliminating the need for repeated manual breeding operations to achieve homozygosity. The system serves itself by using its own components (Cas9 and guide RNA) to propagate the mutation.
Solution Approach 2:
The gene drive allele is designed to perform the mutation and propagation function in advance. By incorporating the CRISPR-Cas9 machinery within the drive allele itself, the system prepares and executes the genotype conversion before traditional breeding would require multiple generations of crossing and selection.
2Manufacturing precision
If traditional breeding methods are used to combine alleles at multiple loci, then compound mutant genotypes can be achieved, but the cost measured in animals, time, and money increases exponentially
Solution Approach 1:
The gene drive system enables self-propagation of desired alleles through the population. The CRISPR-Cas9 mechanism automatically copies the drive allele from one chromosome to its homologous counterpart in the germline, eliminating the need for repeated manual breeding operations to achieve homozygosity. The system serves itself by using its own components (Cas9 and guide RNA) to propagate the mutation.
3Manufacturing precision
If Mendelian crossing is used for each new locus, then desired mutant genotypes can be produced, but generation time and production time increase substantially
Solution Approach 1:
The gene drive system enables self-propagation of desired alleles through the population. The CRISPR-Cas9 mechanism automatically copies the drive allele from one chromosome to its homologous counterpart in the germline, eliminating the need for repeated manual breeding operations to achieve homozygosity. The system serves itself by using its own components (Cas9 and guide RNA) to propagate the mutation.
Solution Approach 2:
The gene drive allele is designed to perform the mutation and propagation function in advance. By incorporating the CRISPR-Cas9 machinery within the drive allele itself, the system prepares and executes the genotype conversion before traditional breeding would require multiple generations of crossing and selection.
4Manufacturing precision
If genetic linkage is present between loci, then recombination is required to combine engineered alleles, but this requires rare recombination events and substantially increases time and animals needed
Solution Approach 1:
The gene drive system enables self-propagation of desired alleles through the population. The CRISPR-Cas9 mechanism automatically copies the drive allele from one chromosome to its homologous counterpart in the germline, eliminating the need for repeated manual breeding operations to achieve homozygosity. The system serves itself by using its own components (Cas9 and guide RNA) to propagate the mutation.
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 reduces the time and cost of producing research and commercial animal models by enabling the rapid generation of desired mutant genotypes, including those for disease modeling and population control, while allowing for the creation of sterile or pesticide-sensitive rodents.
Implementation Method 1
an active NHEJ pathway would be highly mutagenic. Indeed, the molecular mechanisms of non-homologous end joining (NHEJ) are repressed during meiosis in many species, including mice, and homology directed repair (HDR) occurs by inter-homologue rather than inter-sister exchange. However, the frequency of inter-homologue recombination after CRISPR-Cas9 induced DSB formation in the germ line has not yet been measured in a mammal.
Implementation Method 2
A CRISPR-Cas9 mediated gene drive leverages the native cellular mechanism of homology directed repair to copy a desired allele from one chromosome to another. This process can convert a heterozygous genotype to homozygosity in a single generation of any animal, including mammals such as rodents.
Implementation Method 3
A CRISPR-Cas9 mediated gene drive leverages the native cellular mechanism of homology directed repair to copy a desired allele from one chromosome to another.
Implementation Method 4
A CRISPR-Cas9 mediated gene drive leverages the native cellular mechanism of homology directed repair to copy a desired allele from one chromosome to another. This process can convert a heterozygous genotype to homozygosity in a single generation of any animal, including mammals such as rodents.
Data Source
AI summary
Provided are systems, constructs, genetically modified organisms, and methods for creating transgenic rodent research and commercial models of human physiology, disease, syndromes, and disorders. Provided are genetically modified rodents encoding for an sgRNA useful in a Cas9-mediated split gene-drive system for optimization of the gene drive system in rodents.