Dual Strand Nickase Editing via Reverse Transcriptase Fusion
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Solution Overview
Problem
Current nucleic acid-guided nuclease editing methods, such as CRISPR, face challenges in achieving high editing efficiency and precision, particularly in mammalian cells, with low editing rates often in the single digits due to limitations in targeting and the need for double-strand breaks which can introduce errors.
Innovation Solution
The use of nickase/reverse transcriptase fusion enzymes and dual CF editing cassettes that nick opposite DNA strands, allowing for targeted editing without double-strand breaks, utilizing reverse transcriptase to incorporate edits directly into the genome, eliminating the need for donor DNA and reducing error rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional nucleic acid-guided nuclease editing methods are used, then targeted genome editing can be achieved, but editing efficiency remains low (single digits) due to the need for double-strand breaks and limitations in targeting
Solution Approach 1:
The patent divides the editing process into two separate nicking events on opposite DNA strands, each guided by a distinct gRNA. This segmentation allows each nickase-gRNA complex to independently target and nick its specific strand without requiring simultaneous double-strand break formation, thereby improving editing efficiency while maintaining precision and reducing errors associated with traditional nuclease methods
Solution Approach 2:
The patent introduces a protein-primed reverse transcriptase as an intermediary enzyme that uses the nicked DNA strand as a primer to synthesize new DNA incorporating the desired edit. This intermediary mechanism bypasses the need for endogenous mismatch repair systems and donor DNA templates, directly incorporating edits at the nick site with high efficiency and accuracy
2Manufacturing precision
If double-strand breaks are induced for genome editing, then permanent edits can be achieved, but error rates increase and accessibility to certain genomic regions is limited
Solution Approach 1:
The patent applies preliminary anti-action by using nickases instead of nucleases to create single-strand nicks rather than double-strand breaks. This preliminary, less aggressive action prevents the harmful effects of DSBs (chromosomal rearrangements, indels, cell death) while still enabling permanent edits through the subsequent reverse transcriptase-mediated DNA synthesis that incorporates the desired sequence changes
3Adaptability or versatility
If traditional nuclease editing methods are used, then genome editing can be performed, but donor DNA is required which increases complexity and reduces accessibility to inaccessible genomic regions
Solution Approach 1:
The patent implements self-service by designing the system to use the nicked DNA strand itself as the primer for reverse transcriptase-mediated DNA synthesis. The reverse transcriptase uses the 3' hydroxyl group created by the nickase to prime synthesis, eliminating the need for external donor DNA templates. This self-priming mechanism simplifies the editing system and enables access to genomic regions where donor DNA delivery would be problematic
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 editing efficiency and accuracy by enabling the incorporation of complementary edits on both DNA strands, reducing errors and increasing accessibility to previously inaccessible genomic regions, surpassing traditional methods like prime editing.
Implementation Method 1
The reverse transcriptase portion of the nickase-RT fusion uses the repair template(s) of each CF editing cassette to synthesize and edit at nicks created by the nickase(s) on opposite DNA strands of the target locus
Implementation Method 2
nucleic acid-guided nickase editing utilizing gRNAs and/or CFgRNAs to facilitate editing of opposite strands of a target locus
Data Source
AI summary
The present disclosure provides compositions of matter, methods and instruments for nucleic acid-guided nickase/reverse transcriptase fusion enzyme editing of nucleic acids in live mammalian cells.