Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq
Name
acssynbio.0c00299.pdf
Description
Published version
Size
2.78 MB
Format
Unknown
Checksum (MD5)
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Author(s)
Brady, Joseph R.
Tan, Melody C.
Whittaker, Charles A.
Colant, Noelle A.
Dalvie, Neil C.
Love, Kerry Routenberg
Love, J. Christopher
Date Issued
August 2020
Journal
ACS Synthetic Biology
Publisher
American Chemical Society (ACS)
Version
Final published version
Abstract
Copyright © 2020 American Chemical Society. 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.
MIT Department
Koch Institute for Integrative Cancer Research at MIT
Massachusetts Institute of Technology. Department of Chemical Engineering
Terms of Use
Creative Commons Attribution 4.0 International license
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DOI of Published Version
https://doi.org/10.1021/acssynbio.0c00299
