3′-Truncated crRNA for Precise Single-Base Genome Editing
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Solution Overview
Problem
The CRISPR/Cas9 system faces challenges in introducing single base mutations due to mismatch tolerance, leading to inefficient and inaccurate genome editing, particularly with the CRISPR/Cpf1 system, which struggles to target specific sites with high precision.
Innovation Solution
The use of 3′-truncated crRNA, where 1 to 5 nucleotides are deleted from the 3′-end, enhances the CRISPR/Cpf1 system's ability to overcome mismatch tolerance, allowing for precise single-base genome editing by improving the recognition and editing efficiency at the target DNA site.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If the CRISPR/Cpf1 system is used for genome editing, then the ability to target T-rich regions is improved, but mismatch tolerance causes double-strand breaks at non-target sites with point mutations
Solution Approach 1:
The patent applies local quality by modifying only the 3′-end region of the crRNA (truncating 1-5 nucleotides) while keeping the rest of the crRNA sequence intact. This localized modification specifically addresses the mismatch tolerance problem at the target recognition interface without affecting the overall targeting capability of the crRNA-Cpf1 complex
Solution Approach 2:
The patent changes the physical parameter of crRNA length by truncating 1-5 nucleotides from the 3′-end. This parameter change fundamentally alters the binding characteristics of the crRNA-Cpf1 complex, reducing its tolerance for mismatches while preserving its ability to recognize and bind to the intended target sequence
2Productivity
If oligonucleotide-directed mutagenesis is used with CRISPR/Cpf1, then negative selection can identify mutated strains, but single base mutation efficiency remains low due to mismatch tolerance
Solution Approach 1:
The invention applies local quality by specifically modifying the 3′-end region of the crRNA (truncating 1-5 nucleotides) while keeping the rest of the crRNA sequence intact. This localized modification specifically addresses the mismatch tolerance problem at the target recognition interface without affecting the overall targeting capability of the crRNA-Cpf1 complex
Solution Approach 2:
The patent changes the physical parameter of crRNA length by truncating 1-5 nucleotides from the 3′-end. This parameter change fundamentally alters the binding characteristics of the crRNA-Cpf1 complex, reducing its tolerance for mismatches while preserving its ability to recognize and bind to the intended target sequence
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 the efficiency and accuracy of single-base genome editing, enabling precise modifications to the target DNA, as demonstrated by improved editing efficiencies in E. coli and other organisms.
Implementation Method 1
the crRNA and the target DNA are complementary to each other
Data Source
AI summary
The present disclosure relates to a method of editing a genome based on the CRISPR/Cpf1 system and a use thereof, and the CRISPR system using an oligonucleotide-induced mutation and 3′-truncated crRNA according to the present disclosure provides the significant effect of genome editing to the target DNA and thus it is expected that the CRISPR system of the present disclosure may be used in a wide range of fields such as a composition for gene editing using gene scissors, screening at the genome level, therapeutics for various diseases including cancer, development of a composition for disease diagnosis or imaging, and development of transgenic animals and plants.