CRISPR/Cas9 Single-Base Editing via Mismatch Guide RNA
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Solution Overview
Problem
The CRISPR/Cas9 system faces challenges in achieving precise genome editing at a single base level due to mismatch tolerance, which limits its ability to introduce point mutations efficiently, particularly in microorganisms, as it often recognizes and cleaves target sites with minor mismatches, leading to low editing yields and difficulty in obtaining desired mutant strains.
Innovation Solution
Introducing mismatched nucleotides between the guide RNA and target DNA using a donor nucleic acid molecule and mismatch guide RNA, which enhances editing efficiency by preventing recognition of target DNA by the CRISPR system, allowing for accurate single-base point mutations and improved editing outcomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the CRISPR/Cas9 system uses standard guide RNA with high complementarity to target DNA, then the system can recognize and cleave target sites effectively, but it also tolerates mismatches and cleaves sites with minor mutations, reducing editing precision
Solution Approach 1:
The patent modifies the guide RNA to include intentional mismatches at specific positions (particularly at the 3' end near the PAM site) to change the binding characteristics. This parameter change allows the system to distinguish between perfectly matched target sites and sites with mutations, thereby achieving single-base editing precision while maintaining reliable target recognition through the PAM sequence requirement
Solution Approach 2:
The patent introduces a donor oligonucleotide as an intermediary molecule that provides the correct sequence template for repair. This donor molecule mediates the repair process after Cas9 creates the double-strand break, ensuring that the desired single-base mutation is introduced while preventing off-target effects by requiring both the mismatched guide RNA binding and the donor template presence for successful editing
2Productivity
If the CRISPR/Cas9 system is used to introduce point mutations in microorganisms, then genome editing can be achieved, but the mismatch tolerance causes the system to recognize and cleave mutated sites, leading to low editing yields
Solution Approach 1:
The patent performs preliminary action by introducing a donor oligonucleotide into the cell before or simultaneously with Cas9 expression. This donor molecule is pre-positioned at the target site, so when Cas9 creates the double-strand break, the repair machinery immediately uses the donor template to introduce the desired mutation. This preliminary positioning of the donor ensures high editing yield by preventing re-cleavage of mutated sites
Solution Approach 2:
The patent converts the harmful mismatch tolerance of CRISPR/Cas9 into a beneficial feature. By designing guide RNA with intentional mismatches at specific positions, the system is made to tolerate only the desired mismatch pattern while rejecting other mismatches. This converts the general mismatch tolerance problem into a specific advantage for distinguishing wild-type from mutated sequences, thereby improving point mutation introduction efficiency
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 increases genome editing efficiency, enabling precise editing at a single base level with minimal off-target effects, as demonstrated by high editing efficiencies achieved in E. coli strains, facilitating the production of mutant strains with specific mutations.
Implementation Method 1
guide RNA that complementarily bind to the target DNA
Implementation Method 2
generating one or more mismatched nucleotides between the target DNA and the guide RNA sequence by a donor nucleic acid molecule and the guide RNA that complementarily bind to the target DNA
Data Source
AI summary
The present invention pertains to a genome editing method based on a CRISPR/CAS9 system, and a use thereof. A CRISPR system using oligonucleotide-induced mutagenesis and mismatch guide RNA (sgRNA) according to the present invention achieves a significant genome editing effect on target DNA. Thus, it is expected that the CRISPR system of the present invention will be able to be used in a wide range of fields, such as compositions for gene editing using genetic scissors, genome level screening, therapeutic agents for treating various diseases including cancer, the development of compositions for disease diagnosis or imaging, and the development of transgenic plants and animals.