Chloroplast and Mitochondria Cytosine Base Editors in Plants
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Solution Overview
Problem
Current genome editing technologies, such as CRISPR, face challenges in efficiently editing chloroplast and mitochondrial genomes in plants due to difficulties in delivering guide RNA into organelles, and existing methods like mtTALENs have low gene editing efficiency.
Innovation Solution
Development of cytosine base editors using plant-specific targeting peptides and a TALE array protein with a deaminase and uracil glycosylase inhibitor, encoded in DNA vectors, to specifically target and edit chloroplast and mitochondrial genomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-based genome editing is used, then nuclear genome editing is efficient, but organelle genome editing is not feasible due to difficulty in delivering guide RNA into organelles
Solution Approach 1:
The invention extracts and removes the guide RNA component from the CRISPR system, replacing it with a TALE-based DNA binding domain that directly targets the organelle genome without requiring RNA delivery. This eliminates the delivery barrier while maintaining targeted editing capability.
Solution Approach 2:
The TALE array protein serves as an intermediary between the targeting peptide and the deaminase enzyme, enabling specific DNA binding and positioning the deaminase at the correct location for base editing without requiring guide RNA.
2Ease of operation
If mtTALENs are used to target mitochondria, then mitochondrial targeting is achieved, but gene editing efficiency is very low due to inefficient repair of DSBs
Solution Approach 1:
The invention extracts and removes the endonuclease domain from the TALEN system, eliminating double-stranded DNA breaks entirely. Instead, it uses a deaminase enzyme to perform direct base conversion, bypassing the inefficient DSB repair pathway in mitochondria.
Solution Approach 2:
The invention replaces the mechanical DSB creation and repair mechanism with a chemical deamination reaction catalyzed by the deaminase enzyme, which directly converts cytosine to uracil without requiring DNA breakage or complex repair pathways.
3Measurement precision
If DddA-derived cytosine base editors are used in human cells, then C·G to T·A conversions are achieved with high target specificity, but the system is not adapted for plant organelles
Solution Approach 1:
The invention modifies the DddA base editor by adding plant-specific chloroplast or mitochondrial targeting peptides to the N-terminus, enabling the enzyme to localize to plant organelles while maintaining its cytosine deaminase activity and target specificity.
Solution Approach 2:
The invention creates a chimeric protein combining the plant-specific targeting peptide, the TALE array protein for DNA recognition, and the DddA deaminase domain for base editing, resulting in a composite enzyme adapted for plant organelle editing with both specificity and organelle targeting capability.
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 solution achieves high target specificity and efficiency in C·G to T·A conversions in plant chloroplast and mitochondrial DNA, overcoming previous limitations in organelle editing.
Implementation Method 1
DddA-derived cytosine base editors (DdCBEs) that catalyze C·G to T·A conversions
Implementation Method 2
TALEs of bacterial origin recognize DNA sequences of target sites following a TALE DNA recognition code
Implementation Method 3
fused with a mitochondrial transition peptide and an uracil glycosylase inhibitor (UGI)
Data Source
AI summary
The present disclosure is generally directed to gene editing in plant chloroplast and plant mitochondrial double-stranded DNA. Disclosed herein are cytosine base editors tailored for chloroplast and mitochondrial genomes in plants using plant-specific chloroplast and mitochondrial targeting peptides, a TALE, and a DNA deaminase. The systems of the present disclosure include DNA vectors and protocols to use them for gene editing in plants.