Unimolecular Guide Molecule Synthesis via Fragment Annealing
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Solution Overview
Problem
Current methods for synthesizing guide molecules for CRISPR systems are costly and inefficient, particularly for commercial-scale production, and often result in contamination with n-1 and n+1 species due to limitations in chemical synthesis techniques.
Innovation Solution
A method involving pre-annealing of guide fragments followed by cross-linking using reactive groups, such as amine-functionalized fragments with urea-based cross-linking, to form unimolecular guide molecules with improved sequence fidelity and reduced contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If chemical synthesis methods are used to produce guide molecules, then manufacturing cost is reduced and production is simplified, but the guide molecules are contaminated with n-1 and n+1 species
Solution Approach 1:
The guide molecule is divided into multiple separate oligonucleotide fragments that are synthesized individually through chemical synthesis, then assembled together through annealing and crosslinking. This segmentation allows each fragment to be synthesized with high purity while avoiding the accumulation of n-1 and n+1 species that would occur in full-length chemical synthesis
Solution Approach 2:
The oligonucleotide fragments are pre-annealed to form duplexes before the final crosslinking step. This preliminary annealing ensures proper base pairing and structural formation, allowing the fragments to be joined in the correct orientation and reducing misassembly errors
2Manufacturing precision
If in-vitro transcription is used to synthesize guide molecules, then full-length sequences are obtained, but manufacturing is costly and limited in scale
Solution Approach 1:
Instead of transcribing the entire guide molecule sequence in a single in-vitro transcription reaction, the method segments the sequence into multiple shorter oligonucleotide fragments that can be chemically synthesized. These fragments are then assembled to reconstruct the full-length guide molecule, achieving both the scalability of chemical synthesis and the full-length accuracy of in-vitro transcription
Solution Approach 2:
The oligonucleotide fragments serve as intermediaries between the chemical synthesis process and the final guide molecule product. These fragments are chemically synthesized with high purity, then used as building blocks that are annealed and crosslinked to form the complete guide molecule, bridging the gap between synthesis method and final product quality
3Productivity
If T7 polymerase is used for in-vitro transcription, then RNA synthesis occurs, but sequences starting with 5' guanine are transcribed more efficiently than other bases
Solution Approach 1:
The method extracts the problematic 5' end sequence from the transcription process by using chemical synthesis to create oligonucleotide fragments with the desired 5' sequences. This avoids the T7 polymerase bias toward guanine-starting sequences, as the fragments are synthesized chemically with precise control over their 5' ends, then assembled into the complete guide molecule
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
The approach results in high-purity, full-length unimolecular guide molecules with minimal n-1 and n+1 species, enhancing their effectiveness and specificity in genome editing applications.
Implementation Method 1
annealing a first oligonucleotide and a second oligonucleotide to form a duplex between a 3' region of the first oligonucleotide and a 5' region of the second oligonucleotide
Implementation Method 2
conjugating the annealed first and second oligonucleotides via the first and second reactive groups to form a unimolecular guide RNA molecule that includes a covalent bond linking the first and second oligonucleotides
Data Source
AI summary
Chemical syntheses of guide molecules are disclosed, along with compositions and methods relating thereto.