Cas Gene Mutagenesis Using DGR Reverse Transcription Control
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for generating nucleic acid diversity in CRISPR-associated (Cas) genes are limited by low mutagenesis rates, inability to control mutation positions, and high risk of introducing nonsense mutations, particularly when performed in vivo.
Innovation Solution
A method utilizing a mutagenic reverse transcriptase from a Diversity Generating Retroelement (DGR) system, coupled with oligonucleotide recombineering, to introduce targeted mutations in Cas genes in vivo, allowing precise control over mutation positions and reducing nonsense mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If random mutagenesis is used to generate diversity in Cas genes, then a large number of variants can be produced, but the mutagenesis rate is low and many nonsense mutations are introduced
Solution Approach 1:
The invention applies local quality by using a targeted mutagenesis system that introduces mutations only at specific positions within the Cas gene coding sequence. The DGR system with its template region and variable region allows mutations to be confined to desired segments, avoiding random nonsense mutations while maintaining high diversity generation at functional positions.
Solution Approach 2:
The invention changes the parameter of mutagenesis control by using a defined template region with specific adenine positions that determine where mutations occur. By designing the template region with controlled adenine content and positioning, the system achieves predictable mutation rates and positions, eliminating the randomness and low efficiency of traditional mutagenesis.
2Adaptability or versatility
If in vivo mutagenesis is performed to enable continuous evolution, then both diversification and selection can happen in vivo, but control over mutation positions is lost and nonsense mutations increase
Solution Approach 1:
The invention maintains local quality control in the in vivo setting by using a plasmid-based DGR system where the template region is defined within the plasmid sequence. The variable region is targeted to specific positions in the Cas gene, ensuring that even during continuous in vivo evolution, mutations occur only at predetermined locations with controlled properties.
Solution Approach 2:
The invention applies preliminary action by pre-designing the template region with specific adenine positions and sequences before introducing it into the in vivo system. This preliminary design ensures that when the DGR system operates continuously in vivo, mutations are generated at the pre-planned positions with the desired characteristics, maintaining precision throughout the evolution process.
3Manufacturing precision
If traditional error-prone PCR is used for targeted mutagenesis, then mutations can be introduced in a controlled manner, but the process is cumbersome especially when many cycles of evolution are performed
Solution Approach 1:
The invention applies self-service by creating an autonomous in vivo DGR system that performs mutagenesis automatically within the cell. The system uses endogenous cellular machinery for transcription and translation, along with the introduced DGR components, to generate mutations without requiring repeated external PCR interventions. The system self-regulates the mutagenesis process through the defined template and variable regions.
Solution Approach 2:
The invention replaces the mechanical PCR-based mutagenesis system with a biological in vivo system. Instead of using thermal cycling and enzymatic reactions in a test tube, the mutagenesis is performed inside living cells using cellular transcription/translation machinery and the DGR system, simplifying the overall process for continuous evolution applications.
4Manufacturing precision
If DGR system is used to introduce controlled sequence diversity, then mutation positions can be controlled, but the system requires specific recognition sequences that limit applications
Solution Approach 1:
The invention achieves universality by designing the DGR system to work with any Cas gene sequence. The template region can be designed to target any desired coding sequence, and the system does not require specific recognition sequences in the target gene. The modular plasmid-based design allows the same DGR machinery to be applied to different Cas genes and even other target genes, making the system broadly applicable.
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 the generation of a large number of protein sequence variants in a targeted manner, with predictable mutagenesis and low nonsense mutation rates, suitable for directed evolution of Cas proteins and transferable to eukaryotic cells.
Implementation Method 1
a variable region within the genome will be overwritten by a DNA fragment produced from a near repeat template region in a process involving transcription, error-prone reverse transcription of the template and recombination
Implementation Method 2
The error-prone reverse transcription ensures the introduction of genetic diversity at the variable region
Implementation Method 3
recombining the mutagenized cDNA with the homologous DNA sequence of the Cas gene in the recombinant cell
Data Source
AI summary
Provided are methods comprising expressing in a recombinant cell comprising a Cas gene a recombinant error-prone reverse transcriptase (RT) and a recombinant spacer RNA comprising a target sequence for mutagenesis of a DNA sequence in the Cas gene; making a mutagenized cDNA polynucleotide homologous to the DNA sequence in the recombinant cell; expressing a recombinant recombineering system in the recombinant cell; and recombining the mutagenized cDNA with the homologous DNA sequence of the Cas gene in the recombinant cell. Also provided are recombinant cells comprising recombinant coding sequences for a recombinant Cas protein, recombinant error-prone reverse transcriptase (RT), recombinant spacer RNA comprising the target sequence, and recombinant recombineering system.