Brady, JR; Tan, MC; Whittaker, CA; Colant, NA; Dalvie, NC; Love, KR; Love, JC; (2020) Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq. ACS Synthetic Biology , 9 (9) pp. 2515-2524. 10.1021/acssynbio.0c00299.

Preview

Text acssynbio.0c00299.pdf - Published Version Download (2MB) | Preview

Abstract

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.

Download activity - last month

Export as CSVJSONXML

Download activity - last 12 months

Export as XMLJSONCSV

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item