Circularized RNA Expression Vector Library Generation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for generating RNA/DNA-encoded gene perturbation libraries, such as CRISPR/Cas systems, suffer from biases and inefficiencies, particularly when dealing with large vectors or complex libraries, which impede the scale and quality of genetic screening applications.
Innovation Solution
A method for generating covalently closed circularized (ccc) DNA-based small RNA/DNA expression vectors or vector libraries without the need for classical cloning technologies, using a single-stranded phagemid vector and mutagenic RNA or DNA primers to introduce diverse small RNA/DNA sequences, thereby overcoming biases and improving library complexity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If classical cloning technologies are used to generate RNA/DNA-encoded gene perturbation libraries, then the library generation process is well-established and reliable, but biases and inefficiencies occur particularly when dealing with large vectors or complex libraries
Solution Approach 1:
The patent replaces classical mechanical cloning methods (restriction enzymes, ligases, transformation) with an in vitro transcription-based system. RNA primers are synthesized in vitro and used to prime DNA synthesis directly in solution, eliminating the need for bacterial transformation and plasmid propagation steps. This substitution of mechanical/biological cloning machinery with in vitro biochemical reactions resolves the contradiction by maintaining reliability through controlled enzymatic reactions while dramatically improving productivity for large vector libraries.
Solution Approach 2:
The patent introduces RNA primers as intermediaries in the library generation process. These RNA primers serve as mediators that bridge the gap between the desired library sequences and the final DNA vectors. The RNA primers are synthesized in vitro with high precision and then used to prime DNA synthesis, acting as an intermediary that enables efficient library generation without classical cloning steps. This intermediary approach allows for better control over library complexity and reduces biases associated with traditional methods.
2Stability of the object's composition
If classical cloning methods are used for large vectors, then the vector structure is maintained, but sequence mutation becomes difficult and library complexity is limited
Solution Approach 1:
The patent performs preliminary action by synthesizing RNA primers in vitro before the DNA synthesis step. These RNA primers contain the desired sequence variations and are prepared with high precision before being used to prime DNA synthesis. This preliminary preparation of diverse RNA primers allows for extensive sequence adaptability in the final library while maintaining vector structure stability, as the primers are designed to anneal to specific regions of the vector backbone. The in vitro synthesis of multiple primer variants enables comprehensive sequence coverage without requiring repeated cloning operations.
Solution Approach 2:
The patent changes the physical state and chemical composition parameters of the priming material by using RNA instead of DNA primers, and by synthesizing these primers in vitro rather than through bacterial culture. This parameter change enables greater versatility in sequence design, as RNA can be synthesized with higher fidelity and more diverse sequences than traditional DNA cloning methods. The in vitro transcription process allows for precise control over primer sequence, length, and modifications, thereby increasing adaptability while maintaining vector integrity through controlled hybridization and extension reactions.
3Ease of manufacture
If conventional cloning technologies are used, then the process is straightforward for simple libraries, but biases occur and library complexity is reduced
Solution Approach 1:
The patent replaces the complex mechanical system of classical cloning (multiple enzymatic steps, bacterial transformation, colony picking, sequencing verification) with a streamlined in vitro transcription and DNA synthesis system. This substitution maintains ease of manufacture by using commercially available enzymes and reagents in a controlled tube-based protocol, while simultaneously improving manufacturing precision by eliminating stochastic biases associated with bacterial transformation efficiency and colony formation. The in vitro system allows for more accurate and uniform representation of all library members.
4Stability of the object's composition
If classical mutagenesis methods are used on large vectors, then the vector backbone is preserved, but sequence introduction becomes inefficient and time-consuming
Solution Approach 1:
The patent extracts the sequence introduction step from the traditional cloning workflow by using in vitro synthesized RNA primers that directly incorporate the desired sequences. Instead of introducing sequences through time-consuming mutagenesis steps on large vectors, the method extracts and separates the sequence synthesis step (performed in vitro with high efficiency) from the vector backbone maintenance step. The RNA primers carry the new sequences and are incorporated directly during DNA synthesis, preserving vector backbone integrity while dramatically reducing the time required for sequence introduction compared to classical mutagenesis methods.
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 method enables the generation of highly diverse and unbiased RNA/DNA expression vector libraries, suitable for targeted gene knockout, knockdown, and genome modification, without the limitations of conventional cloning methods, thus enhancing the efficiency and accuracy of genetic screening applications.
Implementation Method 1
a first homology region, a target sequence region encoding for a small RNA/DNA to be expressed, and a second homology region, wherein the first homology region is complementary to, or is capable of annealing to, a sequence of the ss-phagemid vector construct
Implementation Method 2
amplifying a covalently closed circularized (ccc)-heteroduplex dsDNA
Data Source
AI summary
The present invention pertains to a novel method for the generation of highly diverse RNA expressing vectors and vector libraries for use in targeted gene knock out, knock down and genome modification approaches. The invention pertains to a method for generating such higher order libraries without the need of classical cloning technologies. This is particularly useful for libraries based on large vectors wherein a sequence cannot be easily mutated with classical mutagenesis methods. The vectors and libraries generated according to the methods of the invention are in particular for RNA assisted silencing technologies such as RNA interference, and for targeted genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system or similar RNA/DNA-encoded gene perturbation systems which use small guide RNAs to target the CRISPR complex to a specific genomic sequence. The invention provides also kits comprising the materials for performing the methods of the invention.