Ribozyme-Containing Yeast Editing Cassettes for Multiplex Genome Engineering
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Solution Overview
Problem
Current nucleic acid-guided nuclease editing systems for yeast cells face limitations in achieving high transcription and nuclear localization of guide RNAs, and they are not efficient for simultaneous combinatorial editing, due to the restricted number of RNA polymerase III promoters and their limitations in expression levels and sequence flexibility.
Innovation Solution
The use of ribozyme-containing editing cassettes with RNA polymerase II promoters, which include self-cleaving ribozymes to prevent nuclear export of gRNAs, allowing for increased expression and nuclear retention, and the implementation of multi-vector transformation techniques to enable simultaneous editing of multiple loci in yeast cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If RNA polymerase III promoters are used to drive gRNA transcription, then gRNA expression levels are improved, but the number of available promoters is limited and sequence flexibility is reduced
Solution Approach 1:
The patent changes the fundamental parameter of promoter type from RNA polymerase III to RNA polymerase II, enabling access to a larger pool of promoters with greater sequence flexibility while maintaining high gRNA expression through optimized Pol II promoter selections and transcriptional enhancement elements
2Adaptability or versatility
If RNA polymerase II promoters are used to drive gRNA transcription, then promoter selection flexibility and expression levels are improved, but gRNAs are exported from the nucleus reducing editing efficiency
Solution Approach 1:
The patent extracts and removes the poly-A tail from gRNAs transcribed by RNA polymerase II through the use of self-cleaving ribozymes, thereby eliminating the nuclear export signal that would otherwise cause gRNA export to the cytoplasm and loss of editing efficiency
Solution Approach 2:
The patent introduces self-cleaving ribozymes as intermediary elements between the RNA polymerase II promoter and the gRNA sequence, which process the transcript to remove the poly-A tail and prevent nuclear export while allowing the gRNA to accumulate in the nucleus for efficient editing
3Manufacturing precision
If current editing systems are used, then one edit can be performed per round, but the architecture does not allow simultaneous combinatorial editing of multiple loci
Solution Approach 1:
The patent creates a universal editing platform where a single RNA polymerase II-based system can simultaneously perform multiple editing functions at different genomic loci by co-transforming multiple editing cassettes, each targeting a different location while sharing the same transcriptional and processing machinery
Solution Approach 2:
The patent segments the editing function into separate co-transformable cassettes, each containing a specific gRNA and donor template pair, allowing independent design and optimization of each editing event while enabling simultaneous delivery and execution of multiple edits in a single experimental round
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 enhances the efficiency of nucleic acid-guided nuclease editing by increasing the number of edits per cell and allowing for multiplex simultaneous editing, overcoming the limitations of existing systems by improving gRNA expression and localization, and enabling multiple edits in a single round of transformation.
Implementation Method 1
a self-cleaving ribozyme is added to the 3′ end of the gRNA transcript. This self-cleaving ribozyme cleaves off the poly-A nuclear export tag.
Implementation Method 2
a self-cleaving ribozyme is also added 5′ of the transcript to cleave off the post-transcriptionally added 5′ cap, which also, when present, helps mediate nuclear export of mRNA
Data Source
AI summary
The present disclosure provides compositions of matter, methods and instruments for editing nucleic acids in live yeast cells.