Uncapped RNA Guide for CRISPR Gene Editing in Yeast
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Solution Overview
Problem
Gene targeting in non-conventional yeast species, such as Yarrowia lipolytica, using RNA-guided endonucleases (RGENs) is challenging due to low efficiency and reliance on non-homologous end-joining (NHEJ) DNA repair processes, making it difficult to achieve scalable and cost-effective modifications.
Innovation Solution
A method involving a non-conventional yeast comprising a CRISPR-associated Cas9 endonuclease and an RNA component without a 5'-cap, utilizing an RNA polymerase II promoter linked to a ribozyme sequence for producing guide RNAs, which form a RNA-guided endonuclease (RGEN) capable of binding and cleaving target sites on chromosomes or episomes, facilitating homologous recombination (HR) and enabling efficient gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If homologous recombination (HR) is used for gene targeting in non-conventional yeast, then gene modification can be achieved, but the efficiency is low and the process is labor-intensive
Solution Approach 1:
The patent introduces RNA-guided endonucleases (RGENs) as intermediary molecules that mediate between the guide RNA and target DNA sequence. The RGEN binds to the guide RNA and uses it to locate the specific target site, where it creates a double-strand break to initiate HR. This intermediary system significantly improves targeting efficiency compared to traditional HR methods that rely on random integration or low-efficiency recombination events.
Solution Approach 2:
The patent replaces the mechanical/manual process of traditional HR gene targeting with an RNA-guided molecular recognition system. Instead of relying on labor-intensive procedures and selectable phenotypes to identify successful targeting events, the system uses programmable guide RNAs to direct RGENs to specific genomic locations, enabling high-throughput, precise gene modification without manual intervention for each target site.
2Manufacturing precision
If traditional HR methods are used, then single gene knock-out can be achieved, but scaling up to multiple genes is difficult and costly
Solution Approach 1:
The patent creates a universal gene editing platform where a single RGEN system can target multiple different genes by simply changing the guide RNA sequence. The Cas9 endonuclease and experimental design are reusable across numerous target sites, allowing researchers to perform multiple gene knock-outs, knock-ins, or modifications using the same core system. This universality dramatically reduces the complexity and cost of multi-gene studies compared to traditional methods that would require separate optimization for each gene.
Solution Approach 2:
The patent enables flexible parameter changes by allowing the guide RNA sequence to be easily modified to target different genomic locations. This parameter change (guide RNA sequence) controls the specificity of the RGEN system, enabling the same Cas9 protein to precisely target different genes without changing the core editing machinery. This flexibility simplifies the process of modifying multiple genes compared to traditional HR methods.
3Duration of action of stationary object
If RNA components with 5'-cap are used in non-conventional yeast, then transcription can be initiated, but the RNA is rapidly degraded and RGEN activity is reduced
Solution Approach 1:
The patent extracts the 5'-cap structure from the guide RNA molecule, creating a uncapped RNA that is resistant to degradation by cellular exonucleases in non-conventional yeast. This extraction of the cap structure eliminates the harmful effect of rapid RNA degradation while preserving the essential functions of guide RNA-mediated target recognition and RGEN binding, thereby improving both RNA stability and RGEN activity.
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 gene editing in non-conventional yeast by promoting HR over NHEJ, allowing for precise modifications and increased throughput in gene targeting, overcoming the limitations of existing methods.
Implementation Method 1
utilizing an RNA polymerase II promoter linked to a ribozyme sequence for producing guide RNAs
Implementation Method 2
the RGEN can bind to the target site sequence. The RGEN can also bind to and cleave the target site
Implementation Method 3
the RNA component and the Cas endonuclease can form a RNA-guided endonuclease (RGEN)
Implementation Method 4
facilitating homologous recombination (HR) and enabling efficient gene editing
Data Source
Figure 1
Figure 2A~2C
Figure 3A~3B
AI summary
Non-conventional yeasts are disclosed herein comprising at least one RNA-guided endonuclease (RGEN) comprising at least one RNA component that does not have a 5'-cap. This uncapped RNA component comprises a sequence complementary to a target site sequence in a chromosome or episome in the yeast. The RGEN can bind to, and optionally cleave, one or both DNA strands at the target site sequence. An example of an RGEN herein is a complex of a Cas9 protein with a guide RNA. A ribozyme is used in certain embodiments to provide an RNA component lacking a 5'-cap. Further disclosed are methods of genetic targeting in non-conventional yeast.