Self-Reconfiguring Genome via CRISPR Logic Gates
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current synthetic biology lacks a means for biological cells to programmatically modify their own DNA, limiting applications such as data logging, logic operations, and self-reconfiguring genomes for synthetic evolution and genomic engineering.
Innovation Solution
A self-reconfiguring genome system based on CRISPR technology, utilizing a cassette with guide RNA, reverse transcriptase, and cleavage enzyme to transcribe and integrate donor RNA into the cellular genome via homologous recombination, enabling serial insertions and reconfiguration of metabolic pathways, as well as cascadable and multiplexable genetic logic gates using RNA as a universal input/output.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If exogenous DNA constructs are introduced into biological cells by conventional means (electroporation, viral packaging, etc.), then genes can be incorporated into the cell genome or maintained as separate entities, but the cell cannot programmatically modify its own DNA
Solution Approach 1:
The patent implements a self-service system where the cell uses its own resources (endogenous RNA polymerase, ribosomes, and cellular machinery) to transcribe guide RNAs, synthesize Cas9 protein, and perform DNA modification. The system leverages the cell's existing biological processes rather than requiring external intervention, enabling programmable self-modification of its own genome.
Solution Approach 2:
The patent creates a universal system where a single Cas9 protein can perform multiple functions: cutting DNA at guide RNA-directed sites, facilitating homologous recombination, and enabling both destructive and constructive genome editing. The endogenous RNA polymerase serves multiple purposes including transcription of guide RNAs and structural RNAs, demonstrating multi-functionality that reduces system complexity.
2Adaptability or versatility
If transcription factor-based logic gates are used to perform logic operations, then genetic circuits can be constructed, but the difficulty of mining or applying directed evolution to find non-interacting recombinases or pairs of chaperone and transcription factor proteins remains
Solution Approach 1:
The patent extracts the logic-gating function from complex protein-protein interaction systems and implements it through simpler RNA-based mechanisms. By using guide RNAs that can be easily designed to target specific DNA sequences, the system achieves logic operations without requiring difficult-to-find non-interacting recombinase or chaperone-transcription factor pairs.
Solution Approach 2:
The patent replaces the mechanical complexity of protein-protein interactions with a more straightforward RNA-DNA hybridization mechanism. Guide RNAs bind to complementary DNA sequences through base pairing, providing a simpler and more predictable basis for logic operations compared to the stochastic protein interactions required in traditional transcription factor-based circuits.
3Adaptability or versatility
If multiple exogenous genes are incorporated to perform complex functions (metabolic pathways, logic functions), then the cell can be programmed for diverse applications, but the process lacks programmable control for self-reconfiguration
Solution Approach 1:
The patent introduces dynamics into the static genome by enabling it to reconfigure itself in response to programmed signals. The cell can dynamically modify its own DNA sequence, add or remove genes, and change its metabolic capabilities through controlled homologous recombination events triggered by guide RNAs and Cas9 protein.
Solution Approach 2:
The system performs preliminary actions by pre-installing the necessary components (guide RNA sequences, Cas9 protein, and homologous recombination machinery) within the cell before the actual genome modification occurs. This allows the cell to autonomously execute complex genetic reconfiguration programs without requiring external intervention at the moment of modification.
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 cells to programmatically modify their DNA, perform logic operations, and reconfigure metabolic pathways, facilitating advanced applications like data logging and synthetic evolution, with enhanced efficiency in genomic modifications and digital logic operations.
Implementation Method 1
utilizing a cassette with guide RNA, reverse transcriptase, and cleavage enzyme to transcribe and integrate donor RNA into the cellular genome
Implementation Method 2
reverse transcriptase, and a cleavage enzyme from the CRISPR system... translating the donorRNA to double-stranded DNA using the reverse transcriptase
Implementation Method 3
recombining the double-stranded DNA via homologous recombination at the cut site of the integration site, thereby producing a genomic modification within the integration site of the cellular genome
Data Source
AI summary
A self-reconfiguring genome uses a cassette having operons or DNA sequences that code for guide RNA, reverse transcriptase, donor RNA, and a CRISPR cleavage enzyme. A self-reconfiguring genome may be based on lambda recombineering of in situ generated oligonucleotides. A method for programmable self-modification of a cellular genome includes transcribing guide RNA from a self-reconfiguring cassette, associating the transcribed guideRNA with the CRISPR enzyme, intercalating a region of complimentary sequence within an integration site of the genome, cutting upstream of a PAM site within the integration site; transcribing the donorRNA, translating donorRNA to double-stranded DNA, and recombining the double-stranded DNA via homologous recombination at the cut site of the integration site. A set of cascadable and multiplexable genetic logic gates with a universal RNA input/output based on single-strand annealing or non-homologous end joining, comprises transcription promoters or terminators, homologous regions, DNA sequences, RNA, and enzymes from the CRISPR system.