Identifying and retargeting transcriptional hot spots in the human genome

Abstract

Mammalian cell line development requires streamlined methodologies that will reduce both the cost and time to identify candidate cell lines. Improvements in site-specific genomic editing techniques can result in flexible, predictable, and robust cell line engineering. However, an outstanding question in the field is the specific site of integration. Here, we seek to identify productive loci within the human genome that will result in stable, high expression of heterologous DNA. Using an unbiased, random integration approach and a green fluorescent reporter construct, we identify ten single-integrant, recombinant human cell lines that exhibit stable, high-level expression. From these cell lines, eight unique corresponding integration loci were identified. These loci are concentrated in non-protein coding regions or intronic regions of protein coding genes. Expression mapping of the surrounding genes reveals minimal disruption of endogenous gene expression. Finally, we demonstrate that targeted de novo integration at one of the identified loci, the 12(th) exon-intron region of the GRIK1 gene on chromosome 21, results in superior expression and stability compared to the standard, illegitimate integration approach at levels approaching 4-fold. The information identified here along with recent advances in site-specific genomic editing techniques can lead to expedited cell line development.

A multi-landing pad DNA integration platform for mammalian cell engineering

“…Interestingly, 20/21 integration sites are found in non-coding regions or gene introns. Similar enrichment of non-coding and intronic regions was observed in transcriptional hot spots in the human genome ( 43 ). Future analysis of a larger set of stable integration sites may help elucidate genomic features that make certain loci better LPs and could also lead to better criteria for identifying prospective stable integration sites in mammalian cells.…”

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology

“…This suggested that they might contain integration events or arrangements that bypass silencing mechanisms. These transformants could provide empirical evidence for putative safe-harbour loci, which have been previously verified in various other organisms including mammalian cell lines (Lee et al, 2015;Cheng et al, 2016;Papapetrou and Schambach, 2016;Salsman and Dellaire, 2016), rice (Cantos et al, 2014) and cyanobacteria (Bentley et al, 2014;Pinto et al, 2015).…”

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

“…Targeted gene integration can improve transgene expression levels when appropriate integration locations are selected; namely, regions that permit insertion of exogenous genes without disrupting the host natural gene expression whilst simultaneously lowering the risk of silencing of the exogenous DNA. Such regions, known as "safe harbours" have been frequently used in human and mouse cell lines [158][159][160]. Identified safe harbour loci knowledge, coupled with efficient endonuclease-mediated targeted integration transformation protocols, can revolutionise genetic engineering strategies to produce recombinant biopharmaceuticals by circumventing reproducibility and stability issues associated with random chromosomal integration [161].…”