CRISPR-Cas9 Editing with Scr7 Inhibitor for Yeast Genome Precision
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Solution Overview
Problem
Current genome engineering technologies, such as CRISPR systems, face challenges with low specificity, high cost, and inefficiency in targeting multiple genomic sites while minimizing off-target effects, particularly in non-conventional yeast cells.
Innovation Solution
Employing a guide RNA/Cas endonuclease system in combination with Scr7, an inhibitor of DNA Ligase IV, to introduce double-strand breaks and enhance Homologous Directed Repair (HDR) while reducing Non-Homologous End Joining (NHEJ) frequency, thereby improving the precision and efficiency of genome editing in non-conventional yeast cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If nuclease-based systems (CRISPR, ZFNs, TALENs) are used to introduce site-specific DNA breaks, then the frequency of homologous recombination at the target locus increases, but off-target activity occurs reducing specificity
Solution Approach 1:
The patent introduces a small molecule inhibitor (SCR7) as an intermediary that specifically blocks DNA Ligase IV activity. This mediator selectively inhibits the NHEJ repair pathway while allowing HDR to proceed, thereby resolving the contradiction between improving HDR frequency and maintaining specificity by preventing off-target effects through selective enzymatic inhibition
Solution Approach 2:
The patent changes the biochemical parameters of the DNA repair process by modulating the activity balance between NHEJ and HDR pathways. By using SCR7 to suppress NHEJ and enhance HDR, the system shifts the repair outcome parameters to favor precise homologous recombination over error-prone non-homologous end joining, thereby improving both productivity and reliability
2Manufacturing precision
If homologous recombination is used for gene knock-out or knock-in, then targeted genome modification is achieved, but the procedure becomes complex and labor-intensive
Solution Approach 1:
The patent extracts and suppresses the complicating NHEJ repair pathway using SCR7 inhibition, leaving the cleaner HDR pathway as the dominant repair mechanism. This extraction of the problematic pathway simplifies the overall procedure by eliminating the need for complex selection markers and screening processes, while maintaining targeted modification precision
Solution Approach 2:
The patent applies preliminary action by pre-treating cells with SCR7 to suppress NHEJ before introducing the nuclease and donor DNA. This preliminary suppression of the competing repair pathway ensures that HDR becomes the predominant repair mechanism, simplifying subsequent steps and reducing the need for complex selection and screening procedures
3Adaptability or versatility
If HR is used for targeting multiple genes in a pathway, then comprehensive genome engineering is achieved, but scalability becomes difficult and costly
Solution Approach 1:
The patent applies universality by using the SCR7 inhibitor in combination with CRISPR-Cas9 systems to create a platform that can target multiple genomic locations simultaneously. The small molecule inhibitor provides a universal mechanism to suppress NHEJ across different cell types and target sites, enabling scalable multi-gene editing without requiring pathway-specific optimization for each target
Solution Approach 2:
The patent replaces the mechanical complexity of traditional HR-based multi-gene targeting with a biochemical solution. By using SCR7 to biochemically suppress NHEJ, the system eliminates the need for complex mechanical procedures such as multiple cloning steps, selectable marker exchanges, and sequential editing, thereby improving scalability and reducing costs
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 frequency of HDR and decreases off-target site effects, allowing for more precise and efficient editing of nucleotides or insertion of polynucleotides into target sites within the genome of non-conventional yeast cells, making the process more scalable and cost-effective.
Implementation Method 1
CRISPR-associated (Cas) RNA-guided endonuclease systems have been developed as a means for introducing site-specific DNA strand breaks at specific target sites
Implementation Method 2
employing a guide RNA/Cas endonuclease system in combination with Scr7, an inhibitor of DNA Ligase IV, to introduce double-strand breaks and enhance Homologous Directed Repair (HDR) while reducing Non-Homologous End Joining (NHEJ) frequency
Data Source
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AI summary
Compositions and methods are provided for editing nucleotides or altering target sites in the genome of a cell. The methods and compositions employ a guide RNA/Cas endonuclease system and at least one component selected from the group consisting of (i) an inhibitor of end joining (NHEJ), (ii) an activator of homology-directed repair (HDR) or (iii) any one combination of (i) and (ii), to provide an effective system for editing nucleotides or altering target sites within the genome of a cell. The present disclosure also describes methods for editing a nucleotide sequence in the genome of a microbial cell employing a guide RNA/Cas endonuclease system and at least one component selected from the group consisting of (i) an inhibitor of NHEJ, (ii) an activator of HDR, or (iii) any one combination of (i) and (ii), wherein said microbial cell has reduced or no off-target site effects.