Identifying Improved Sites for Heterologous Gene Integration Using ATAC-seq

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.

Synthetic Biology Toolkit for Marker-Less Integration of Multigene Pathways into Pichia pastoris via CRISPR/Cas9

“…pastoris. More specifically, a series of integration sites allowing efficient and stable expression of heterologous genes are demanded for extensive metabolic pathway engineering . Therefore, the present study aimed to establish a CRISPR-based synthetic biology toolkit, with a focus on the characterization of potential integration sites, to assemble multigene biosynthetic pathways.…”

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae

“…Similar location effects on heterologous gene expression have been previously observed in S. cerevisiae and Komagataella phaffii. 42,43 The epigenetic effect and enhancer-like effect from surrounding sequences may influence transgene expression. 42 The pIGFP-NES integration through the GAL1 promoter resulted in the P GAL7 -yEGFP-NES cassette being surrounded by GAL promoter-controlled expression cassettes (Figure 6A).…”

Identification of Genomic Safe Harbors in the Anhydrobiotic Cell Line, Pv11

“…Such phenotypic changes are a major concern, especially in human gene therapy applications [ 1 ]. In general, global gene expression patterns are assessed to confirm that the likelihood of any potential phenotypic changes in the future is low [ 3 , 10 ]. In other words, the most important point is whether the intrinsic cellular phenotypes can be maintained after transgenesis.…”

“…In contrast, site-specific knock-in results in more controlled outcomes and has been facilitated by recent advances in genome-editing technologies. Furthermore, in some species and cell lines, several research groups have developed genomic sites called genomic safe harbors (GSHs) that enable transgenes to function as designed without adverse effects on the host [ 1 , 2 , 3 , 4 , 5 , 6 ]. Transgene integration into such GSHs has promoted fundamental and applied biological research, but identifying GSHs is still challenging, especially in non-model species, owing to insufficient development of the genome database and genetic engineering tools.…”