PASTE Genome Editing via Nickase and Reverse Transcriptase
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Solution Overview
Problem
Current genome editing tools, such as CRISPR-Cas systems, rely on cellular repair mechanisms which are error-prone and inefficient for precise gene integration, limiting the effectiveness of programmable gene editing and delivery.
Innovation Solution
The method involves using a DNA binding nuclease with nickase activity linked to a reverse transcriptase domain, guided by a guide RNA to target specific genomic locations for site-specific integration of nucleic acids, enabling precise incorporation of nucleic acids into the genome through integration enzymes like Cre or Bxb1 integrase.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If CRISPR-Cas systems are used for genome editing, then programmable DNA interference is achieved with minimal requirements, but the process relies on error-prone cellular repair mechanisms that limit precision
Solution Approach 1:
The system divides the integration process into two independent modules: (1) a DNA binding nuclease with nickase activity that creates a nick at the target site, and (2) a reverse transcriptase domain that performs site-specific integration of the nucleic acid. This segmentation allows each module to perform its function optimally without relying on error-prone cellular repair mechanisms.
Solution Approach 2:
The patent introduces a guide RNA as an intermediary that directs the DNA binding nuclease to the specific genomic location. The guide RNA mediates between the programmable element and the target DNA, enabling precise targeting while the reverse transcriptase domain ensures accurate integration at the targeted site.
2Reliability
If cellular repair mechanisms are used for DNA repair, then DNA double strand breaks are repaired, but the process is error-prone and generates random indels causing frame shift mutations
Solution Approach 1:
The patent replaces the error-prone cellular repair mechanisms (NHEJ and HR) with an engineered reverse transcriptase domain that performs site-specific integration. This substitution eliminates reliance on the cell's natural repair pathways and provides controlled, precise integration at the targeted genomic location.
Solution Approach 2:
The system changes the fundamental parameter of DNA repair from stochastic cellular processes to a programmable enzymatic reaction. The reverse transcriptase domain uses a template-guided mechanism to ensure accurate integration, transforming the repair process from error-prone to highly precise.
3Manufacturing precision
If homology-directed repair is enhanced by fusion proteins or overlapping homology arms, then precise repairing capability is improved, but the process still relies on host DNA repair systems
Solution Approach 1:
The patent extracts the integration function from the host DNA repair systems entirely. By using a DNA binding nuclease with nickase activity combined with a reverse transcriptase domain, the system performs site-specific integration independently of cellular repair pathways, eliminating the need for complex fusion proteins or overlapping homology arms.
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 allows for efficient and precise site-specific integration of nucleic acids into the genome, reducing errors and improving the accuracy of gene editing, facilitating the treatment of diseases and diagnostics.
Implementation Method 1
the reverse transcriptase domain incorporates the integration sequence of the gRNA into the nicked site, thereby providing the integration site at the desired location of the cell genome
Implementation Method 2
the DNA binding nuclease nicks a strand of the cell genome
Implementation Method 3
the integration enzyme incorporates the nucleic acid into the cell genome at the integration site by integration, recombination, or reverse transcription
Implementation Method 4
the integration enzyme incorporates the nucleic acid into the cell genome at the integration site by integration, recombination, or reverse transcription
Data Source
AI summary
This disclosure provides systems, methods, and compositions for site-specific genetic engineering using Programmable Addition via Site-Specific Targeting Elements (PASTE). PASTE comprises the addition of an integration site into a target genome followed by the insertion of one or more genes of interest or one or more nucleic acid sequences of interest at the site. PASTE combines gene editing technologies and integrase technologies to achieve unidirectional incorporation of genes in a genome for the treatment of diseases and diagnosis of disease.