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Identifying improved sites for heterologous gene integration using ATAC-seq

ABSTRACT: Identifying improved sites for heterologous gene integration using ATAC-seq

PROVIDER: PRJNA645901 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12211226_1.fastq.gz	Fastqsanger.gz
	SRR12211226_2.fastq.gz	Fastqsanger.gz
	SRR12211227_1.fastq.gz	Fastqsanger.gz
	SRR12211227_2.fastq.gz	Fastqsanger.gz
	SRR12211228_1.fastq.gz	Fastqsanger.gz

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Identifying improved sites for heterologous gene integration using ATAC-seq

Project description:We analyzed the chromatin accessibility and nucleosome positioning by ATAC-seq of both null and transgene-expressing strains of Komagataella phaffii (Pichia pastoris) under different growth conditions. These data enabled identification of the features that determine performance of various integration sites for transgene expression. Understanding chromatin accessibility and nucleosome positioning can provide further clarity into gene regulation and expression broadly in this organism.

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Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq.

Project description:Constructing efficient cellular factories often requires integration of heterologous pathways for synthesis of novel compounds and improved cellular productivity. Few genomic sites are routinely used, however, for efficient integration and expression of heterologous genes, especially in nonmodel hosts. Here, a data-guided framework for informing suitable integration sites for heterologous genes based on ATAC-seq was developed in the nonmodel yeast Komagataella phaffii. Single-copy GFP constructs were integrated using CRISPR/Cas9 into 38 intergenic regions (IGRs) to evaluate the effects of IGR size, intensity of ATAC-seq peaks, and orientation and expression of adjacent genes. Only the intensity of accessibility peaks was observed to have a significant effect, with higher expression observed from IGRs with low- to moderate-intensity peaks than from high-intensity peaks. This effect diminished for tandem, multicopy integrations, suggesting that the additional copies of exogenous sequence buffered the transcriptional unit of the transgene against effects from endogenous sequence context. The approach developed from these results should provide a basis for nominating suitable IGRs in other eukaryotic hosts from an annotated genome and ATAC-seq data.

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