Deciphering integration loci of CHO manufacturing cell lines using long read nanopore sequencing

Clappier, Christian; Böttner, Dennis; Heinzelmann, Daniel; Stadermann, Anna; Schulz, Patrick; Schmidt, Moritz J.; Lindner, Benjamin

doi:10.1016/j.nbt.2023.03.003

Cited by 5 publications

(1 citation statement)

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“…For the identification of transgene ISs, it would be, therefore, sufficient to apply nCats with crRNAs that direct the sequencing toward the 5′-junction site (JS) and the 3′-JS of transgenes with the neighboring chromatin (van Haasteren et al, 2021;Hertel et al, 2022). In a subsequent nCats run, crRNAs can be designed that allow to sequence from the genome into the integrated transgene sequence, thus resolving the integrity and arrangement of the EV (Clappier et al, 2023). Apart from identifying or verifying transgene integration, the technique can also be used to specifically assess the epigenetic status of the EV and neighboring endogenous genes at the IS.…”

Section: Introductionmentioning

confidence: 99%

Nanopore Cas9‐targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status in recombinant Chinese hamster ovary cells

Leitner

Motheramgari

Borth

et al. 2023

Biotech & Bioengineering

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The integration of a transgene expression construct into the host genome is the initial step for the generation of recombinant cell lines used for biopharmaceutical production. The stability and level of recombinant gene expression in Chinese hamster ovary (CHO) can be correlated to the copy number, its integration site as well as the epigenetic context of the transgene vector. Also, undesired integration events, such as concatemers, truncated, and inverted vector repeats, are impacting the stability of recombinant cell lines. Thus, to characterize cell clones and to isolate the most promising candidates, it is crucial to obtain information on the site of integration, the structure of integrated sequence and the epigenetic status. Current sequencing techniques allow to gather this information separately but do not offer a comprehensive and simultaneous resolution. In this study, we present a fast and robust nanopore Cas9‐targeted sequencing (nCats) pipeline to identify integration sites, the composition of the integrated sequence as well as its DNA methylation status in CHO cells that can be obtained simultaneously from the same sequencing run. A Cas9‐enrichment step during library preparation enables targeted and directional nanopore sequencing with up to 724× median on‐target coverage and up to 153 kb long reads. The data generated by nCats provides sensitive, detailed, and correct information on the transgene integration sites and the expression vector structure, which could only be partly produced by traditional Targeted Locus Amplification‐seq data. Moreover, with nCats the DNA methylation status can be analyzed from the same raw data without prior DNA amplification.

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Section: Introductionmentioning

confidence: 99%