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Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [sRNA-Seq]

ABSTRACT: Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [sRNA-Seq]

PROVIDER: PRJNA1213156 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR32054654.fastq.gz	Fastqsanger.gz
	SRR32054655.fastq.gz	Fastqsanger.gz
	SRR32054656.fastq.gz	Fastqsanger.gz
	SRR32054657.fastq.gz	Fastqsanger.gz
	SRR32054658.fastq.gz	Fastqsanger.gz

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Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [sRNA-Seq]

Project description:Improvement and research of plants depends on the long-term expression of transgenes. However, the durability of transgene expression is routinely hampered by silencing pathways that start as the post-transcriptional process of transcript cleavage by RNA interference (RNAi). To avoid transgene silencing, we aimed to inhibit the sorting of transgene mRNAs into RNAi. We manipulated a well-studied protein / RNA-binding module from Arabidopsis into a transgene transcript, where the transcript is now bound by an engineered RNA-binding protein that preferentially sorts the RNA into translation, rather than RNAi. We used the Cas9 transcript as a proof-of-principle and demonstrated higher Cas9 protein production and editing rates. In addition, transgenes with the engineered protein / RNA-binding module had improved long-term durability of transgene expression, as after four generations these plants had higher Cas9 protein accumulation and lower levels of DNA methylation, a hallmark of transgene silencing. Our engineered system represents a successful manipulation of post-transcriptional RNA sorting for improved transgene performance, and could be applied to any transgene transcript.

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Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [BSAS]

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Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [BSAS]

Project description:Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [BSAS]

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GC-rich coding sequences reduce transposon-like sRNA-mediated transgene silencing in Arabidopsis and maize

Project description:The molecular basis of transgene susceptibility to silencing is poorly characterized in plants, thus we evaluated several transgene design parameters as means to reduce heritable sRNA-mediated transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposable element (TE)-like silencing of canola-biased PUFA transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three Bacillus thuringiensis (Bt) crystal proteins tested the impact of CDSs recoding using different codon bias tables. Transgenes with the higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

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