CRISPR/Cas9 Unnatural Nucleotide Retention
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Solution Overview
Problem
The natural genetic alphabet's limited chemical and physical diversity restricts the applications of nucleic acid technologies, such as PCR and isothermal amplification, in biotechnology, particularly in the retention and production of unnatural nucleotides within cells.
Innovation Solution
The use of a CRISPR/Cas editing system, including a Cas9 polypeptide and single guide RNA (sgRNA) with a crRNA-tracrRNA scaffold, to recognize and modify unnatural nucleotides within nucleic acid molecules, modulating their replication and retention in cells, thereby increasing their production and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If polymerases are used to sequence-specifically synthesize/amplify oligonucleotides, then productivity is improved, but the limited chemical/physical diversity of natural nucleotides restricts application versatility
Solution Approach 1:
The patent expands the chemical parameter space of the genetic alphabet by incorporating unnatural nucleotides with modified base structures (e.g., 5-bromouracil, 5-fluorouracil, 2-aminoadenine) that differ from natural A, C, G, T nucleotides. This allows polymerases to synthesize oligonucleotides with enhanced chemical diversity while maintaining synthesis efficiency through selected polymerases capable of incorporating these unnatural nucleotides.
2Adaptability or versatility
If unnatural nucleotides are introduced into nucleic acid molecules, then chemical diversity is improved, but retention and production of these molecules in cells deteriorates
Solution Approach 1:
The patent employs CRISPR/Cas9 systems with specifically designed guide RNAs that recognize and target nucleic acid molecules containing unnatural nucleotides. This creates a feedback mechanism where the cellular machinery actively monitors and enriches for molecules with the desired unnatural nucleotide incorporation, thereby improving retention despite the inherent instability of unnatural nucleotides in biological systems.
Solution Approach 2:
The patent uses CRISPR/Cas9 as an intermediary system that bridges the gap between introducing unnatural nucleotides and achieving their stable retention. The guide RNA acts as a mediator that specifically recognizes the unnatural nucleotide configuration and directs the Cas9 protein to protect or enrich these molecules, enabling reliable cellular retention of chemically diverse nucleic acids.
3Reliability
If CRISPR/Cas9 system is used to recognize and modify unnatural nucleotides, then retention of unnatural nucleotides is improved, but device complexity increases
Solution Approach 1:
The patent leverages the universal CRISPR/Cas9 platform, originally developed for gene editing, and adapts it for the specific function of recognizing and retaining unnatural nucleotides. By designing guide RNAs with modified sequences that complement the unnatural nucleotide configurations, the existing Cas9 protein machinery is repurposed to serve multiple functions: both traditional gene targeting and the new function of enriching/retaining unnatural nucleotide-containing molecules, thereby managing complexity through multi-functionality.
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 enhances the production and retention of nucleic acid molecules containing unnatural nucleotides, improving their stability and availability for applications in biotechnology, including protein synthesis and therapeutic development.
Implementation Method 1
the sgRNA encoded by the second nucleic acid molecule comprises a target motif that recognizes a modification at the unnatural nucleotide position within the third nucleic acid molecule
Implementation Method 2
the Cas9 polypeptide or variants thereof generate a double-stranded break
Data Source
AI summary
Disclosed herein are methods, cells, engineered microorganisms, and kits for increased production of a nucleic acid molecule that comprises an unnatural nucleotide.