Cascade-Deaminase Complex for Base Editing Without DNA Cleavage
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Solution Overview
Problem
Conventional genome editing techniques require double-stranded DNA breaks, leading to cytotoxicity, chromosomal rearrangements, and low mutation efficiency, particularly in primate ova and unicellular microorganisms, and lack effectiveness in gene therapy and molecular breeding due to reliance on cleavage-based methods.
Innovation Solution
A method utilizing a Type I CRISPR-Cas system, specifically the Cascade complex from Escherichia coli, combined with a deaminase to convert nucleotides without cleaving DNA, allowing for targeted nucleotide modifications by recognizing specific PAM sequences, thereby expanding mutation introduction sites beyond the limitations of Type II CRISPR-Cas9.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional genome editing techniques using artificial nucleases are used, then sequence-specific DNA cleavage is achieved, but double-stranded DNA breaks cause cytotoxicity, chromosomal rearrangement, and reduced reliability
Solution Approach 1:
The invention extracts and removes the harmful nuclease domain from the CRISPR-Cas9 system, retaining only the sequence-specific recognition capability through guide RNA and Cas protein binding, while eliminating the DNA cleavage function that causes cytotoxicity and chromosomal rearrangement
Solution Approach 2:
The invention introduces a deaminase enzyme as an intermediary that performs base conversion instead of direct DNA cleavage. The deaminase acts as a mediator between the sequence-specific recognition complex and the DNA, enabling precise nucleotide modification without causing double-stranded breaks
2Reliability
If Type II CRISPR-Cas9 system is used, then DNA cleavage capability is achieved, but mutation introduction sites are limited by PAM sequence requirements
Solution Approach 1:
The invention removes the nuclease activity requirement from the system, allowing the CRISPR-Cas complex to function solely for sequence recognition and guiding deaminase to target sites without being constrained by PAM sequence requirements for cleavage
Solution Approach 2:
The invention changes the fundamental parameter of the editing mechanism from cleavage-based to base conversion-based, which alters the targeting constraints and enables mutation introduction at sites that would not be accessible to traditional Cas9 nucleases
3Reliability
If deaminase alone is used without CRISPR system, then base conversion capability is achieved, but sequence-specific targeting is lost
Solution Approach 1:
The invention merges the sequence-specific recognition capability of the CRISPR-Cas complex with the base conversion capability of deaminase, creating a unified system that combines targeting precision with editing functionality without requiring DNA cleavage
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 safety by avoiding foreign DNA insertion and DNA breaks, increasing mutation efficiency and site flexibility, making it suitable for a wide range of biological materials and reducing toxicity, thus addressing the limitations of existing genome editing methods.
Implementation Method 1
deaminase that converts an amino group of a nucleic acid base to a carbonyl group
Data Source
Figure 1A~1E
Figure 2
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AI summary
The present invention provides a method of modifying a targeted site of a double stranded DNA in a host cell, the method including introducing (a) a DNA encoding a crRNA containing a sequence complementary to a target strand of a target nucleotide sequence in the given double stranded DNA, and (b) a DNA encoding a protein group constituting Cascade and a nucleic acid base converting enzyme, in which the nucleic acid base converting enzyme is constituted in a form capable of forming a complex with any protein in the protein group, into the host cell to convert one or more nucleotides in the targeted site to other one or more nucleotides, or delete one or more nucleotides, or insert one or more nucleotides into said targeted site, without cleaving the double stranded DNA in the targeted site.