RT Editing Compositions Using Cas9 Nickase–Reverse Transcriptase
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Solution Overview
Problem
Existing gene editing approaches suffer from insufficient expression levels, inadequate efficacy, and lack of specificity, making them ineffective for therapeutic applications and in vivo use.
Innovation Solution
A reverse transcriptase (RT)-based editing system comprising a fusion protein with a Cas9 nickase and a template armed guide RNA (tagRNA) for precise nucleotide editing, utilizing a DNA binding domain, endonuclease domain, and polymerase domain to incorporate intended edits into double-stranded DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing gene editing approaches are used, then the editing process can be performed, but the expression levels are insufficient and efficacy is inadequate
Solution Approach 1:
The patent combines multiple functional domains into a single fusion protein: a DNA binding domain (Cas9 nickase), a DNA endonuclease domain, and a DNA polymerase domain (reverse transcriptase). This merging allows the editor to simultaneously perform target recognition, DNA cleavage, and template-directed repair in one molecular complex, thereby achieving sufficient expression levels and therapeutic efficacy without requiring separate delivery of multiple components.
Solution Approach 2:
The fusion protein is designed with multi-functionality, where a single protein molecule performs multiple functions: sequence-specific DNA binding via the Cas9 nickase domain, site-specific nicking via the endonuclease domain, and template-directed DNA synthesis via the reverse transcriptase domain. This multi-functional design ensures reliable editing efficacy while maintaining adequate expression levels.
2Manufacturing precision
If existing gene editing approaches are used, then the editing process can be performed, but there is a lack of specificity in directing a precise editing outcome
Solution Approach 1:
The patent employs local quality by using a guide RNA with a spacer sequence that is complementary to a specific target sequence in the genome. This allows the DNA binding domain to recognize and bind only to the intended target locus, ensuring high editing specificity. The local complementarity between the guide RNA spacer and the target DNA sequence provides precise addressing to the correct genomic location.
Solution Approach 2:
By merging the DNA binding domain with the endonuclease and polymerase domains in a single fusion protein, the patent ensures that all editing activities occur at the same genomic location where the guide RNA directs binding. This spatial coupling prevents off-target effects and enhances specificity while maintaining reliable editing efficacy at the intended target.
3Duration of action of stationary object
If existing gene editing approaches are used, then the editing process can be performed, but the duration of component expression is insufficient for therapeutic effectiveness
Solution Approach 1:
The patent delivers the fusion protein and guide RNA as pre-assembled ribonucleoprotein (RNP) complexes or expresses them from codon-optimized nucleic acid sequences that are designed for sustained expression. The guide RNA includes structural elements that stabilize the complex and extend its half-life in the target cell, ensuring prolonged expression duration necessary for therapeutic effectiveness.
Solution Approach 2:
The patent optimizes parameters including codon usage in the nucleic acid sequences encoding the fusion protein and guide RNA to enhance stability and duration of expression. The guide RNA scaffold is designed with modified structures that increase resistance to degradation, thereby extending the duration of action while maintaining reliable therapeutic effectiveness.
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 RT-based system achieves enhanced editing efficiency and specificity, with nucleotide edit incorporation rates greater than 30-80%, effectively correcting mutations in target DNA sequences.
Implementation Method 1
a reverse transcriptase (RT) editor comprising a DNA binding domain, a DNA endonuclease domain and a DNA polymerase domain... wherein the DNA polymerase domain comprises a reverse transcriptase
Implementation Method 2
a spacer sequence, a scaffold sequence, an editing template... wherein the tagRNA directs the RT editor to incorporate the intended nucleotide edit in the double stranded target DNA
Data Source
AI summary
Disclosed herein include methods, compositions, and kits suitable for use in gene editing. RT editing systems are provided herein comprising a fusion protein comprising a Cas9 nickase and a reverse transcriptase, and a template armed guide RNA (tagRNA) comprising from 5′ to 3′ a spacer sequence, a scaffold sequence, an editing template and a flap binding sequence. In some embodiments, the RT editing system further comprises an enhancer guide RNA (egRNA).