Single-Strand Base Editor for Mitochondrial and Chloroplast DNA
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Solution Overview
Problem
Existing base editors, such as DdCBE and TALED systems, are limited by their inability to perform single-strand-specific editing in nuclear, mitochondrial, and chloroplast DNA, leading to safety concerns, off-target mutations, and lower product purity.
Innovation Solution
A novel nucleic acid base editor comprising a sequence-specific DNA binding protein, a nickase, an exonuclease, and a base-specific deaminase, optionally with a uracil glycosylase inhibitor, which can function in these organelles and achieve high-purity editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas9 base editing is used to achieve targeted base substitution, then editing precision is improved, but the system cannot specifically target single strands in mitochondrial and chloroplast DNA leading to off-target mutations
Solution Approach 1:
The base editor is divided into multiple functional modules: a sequence-specific DNA binding protein (e.g., TALE) for target recognition, a nickase (e.g., FokI nickase) for single-strand nicking, an exonuclease for strand removal, and a base-specific deaminase for base conversion. This segmentation allows each component to perform its specific function independently, enabling precise single-strand targeting in mitochondrial and chloroplast DNA while avoiding off-target mutations.
Solution Approach 2:
The patent introduces an exonuclease as an intermediary component that removes the nicked DNA strand after nicking, creating a single-stranded gap that allows the base-specific deaminase to act specifically on the remaining strand. This intermediary step ensures that base editing occurs only on the intended single strand, preventing off-target effects on the complementary strand.
2Adaptability or versatility
If base editing is expanded to mitochondrial and chloroplast genomes, then application range is improved, but off-target mutations and impure editing products increase
Solution Approach 1:
The base editor system is designed with universal components that can function across different cellular compartments (nucleus, mitochondria, and chloroplasts). The sequence-specific DNA binding protein can be programmed to recognize different target sequences, while the nickase, exonuclease, and deaminase components maintain their functions across these different environments, enabling broad application while maintaining precision through the single-strand-specific mechanism.
Solution Approach 2:
The patent applies local quality by making the editing system strand-specific at the molecular level. The nickase introduces nicks only on one strand, and the exonuclease removes only the nicked strand, creating a localized single-stranded region where base editing occurs. This local strand-specific action ensures high editing product purity by preventing simultaneous editing of both strands, which would lead to impure products and off-target mutations.
3Manufacturing precision
If CRISPR-Cas9 system is used for base editing, then base substitution is achieved, but sgRNA cannot be efficiently transferred into mitochondria and chloroplasts
Solution Approach 1:
The patent extracts the guide RNA component from the CRISPR-Cas9 system and replaces it with a sequence-specific DNA binding protein (e.g., TALE protein). This extraction eliminates the dependency on sgRNA, which cannot efficiently enter mitochondria and chloroplasts. The TALE protein, being a protein rather than RNA, can be efficiently delivered to these organelles while maintaining the ability to specifically recognize and bind target DNA sequences, thus preserving base substitution capability while improving organelle targeting 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
The novel base editor ensures precise, single-strand-specific editing with low off-target rates and high product purity, enhancing safety and efficiency.
Implementation Method 1
a sequence-specific DNA binding protein...binds to the target DNA sequence
Implementation Method 2
a nickase...nicks one DNA strand preferentially at the target site
Implementation Method 3
an exonuclease...degrades the nicked DNA strand from the nick to the binding site
Implementation Method 4
a base-specific deaminase...catalyzes deamination of cytidine or adenosine...converting a C:G base pair to a T:A base pair or an A:T base pair to a G:C base pair
Implementation Method 5
a uracil glycosylase inhibitor...protecting uracil in DNA from being excised
Data Source
AI summary
The present disclosure discloses a base editor and the use thereof. The present disclosure provides a nucleic acid base editor, specifically a base editor which is not based on CRISPR technology. The base editor comprises a sequence-specific DNA binding protein, a nickase, an exonuclease and a base-specific deaminase. This base editor is single-strand-specific, and as compared with conventional base editors, the base editor of the present disclosure has wide applicability in cells and is capable of functioning in the nucleus as well as in mitochondrial DNA and/or chloroplast DNA. This base editor has the characteristics of achieving base editing products with high purity and resulting in few indel byproducts while realizing efficient base editing, which is conducive to being used as an efficient and safe gene editing tool.