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Precision RNA Base Editing in Plant Organelles Using Engineered Synthetic P-type PPR Editing Factors

ABSTRACT: Precision RNA Base Editing in Plant Organelles Using Engineered Synthetic P-type PPR Editing Factors

PROVIDER: PRJNA1155685 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR30511825_1.fastq.gz	Fastqsanger.gz
	SRR30511825_2.fastq.gz	Fastqsanger.gz
	SRR30511826_1.fastq.gz	Fastqsanger.gz
	SRR30511826_2.fastq.gz	Fastqsanger.gz
	SRR30511827_1.fastq.gz	Fastqsanger.gz

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Precision RNA Base Editing in Plant Organelles Using Engineered Synthetic P-type PPR Editing Factors

Project description:RNA editing, particularly cytidine-to-uridine conversions in plant organelles, plays a crucial role in regulating gene expression. While natural PLS-type PPR proteins are specialized in this process, synthetic PPR proteins offer significant potential for targeted RNA editing. In this study, we engineered chimeric editing factors by fusing synthetic P-type PPR guides with the DYW cytidine deaminase domain from a moss mitochondrial editing factor, PPR56. These designer PPR editors (dPPRe) were tested in Escherichia coli and Nicotiana benthamiana chloroplasts and mitochondria, demonstrating efficient and precise de novo RNA editing. Transcriptome-wide analysis of the most efficient chloroplastic dPPRe revealed minimal off-target effects, with only three non-target C sites edited due to sequence similarity with the intended target. This study introduces a novel and precise method for RNA base editing in plant organelles, paving the way for new approaches in gene regulation applicable to plants and potentially other organisms.

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Project description:Programmable base editing of RNA enables rewriting the genetic codes on specific sites. Current tools for specific RNA editing dependent on the assembly or recruitment of the guide RNA into an RNA/protein complex, which may cause delivery barrier and low editing efficiency. Here we report a new set of tools, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. The REWIRE system contains a human-originated programmable RNA-binding domain (PUF domain) to specifically recognize target sequence and different deaminase domains to achieve A-to-I or C-to-U editing. By utilizing this system, we have achieved editing efficiencies up to 80% in A-to-I editing and 65% in C-to-U editing, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. We applied the REWIREs to correct disease-associated mutations and modify mitochondrial RNAs, and further optimized the REWIREs to improve the editing efficiency and minimize off-target effects. As a single-component base editing system originated from human proteins, REWIRE presents a precise and efficient RNA-editing platform with broad applicability in basic research and gene therapy.

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