How To Train Your Cell - Towards Controlling Phenotypes by Harnessing The Epigenome of Chinese Hamster Ovary Production Cell Lines

Biotechnology Advances 56 (2022) 107924
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Biotechnology Advances
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Research review paper
How to train your cell - Towards controlling phenotypes by harnessing the

epigenome of Chinese hamster ovary production cell lines
Nicolas Marx a, Peter Eisenhut b, Marcus Weinguny a, b, Gerald Klanert b, Nicole Borth a, b, *
a
University of Natural Resources and Life Sciences, Vienna, Austria
b
Austrian Centre for Industrial Biotechnology GmbH, Vienna, Austria
A R T I C L E I N F O A B S T R A C T
Keywords: Recent advances in omics technologies and the broad availability of big datasets have revolutionized our un
Chinese hamster ovary cells derstanding of Chinese hamster ovary cells in their role as the most prevalent host for production of complex
CHO biopharmaceuticals. In consequence, our perception of this “workhorse of the biopharmaceutical industry” has
Phenotype
successively shifted from that of a nicely working, but unknown recombinant protein producing black box to a
Cell line engineering
biological system governed by multiple complex regulatory layers that might possibly be harnessed and
Epigenome
epigenetic modificaitons manipulated at will. Despite the tremendous progress that has been made to characterize CHO cells on various
omics levels, our understanding is still far from complete. The well-known inherent genetic plasticity of any
immortalized and rapidly dividing cell line also characterizes CHO cells and can lead to problematic instability of
recombinant protein production. While the high mutational frequency has been a focus of CHO cell research for
decades, the impact of epigenetics and its role in differential gene expression has only recently been addressed. In
this review we provide an overview about the current understanding of epigenetic regulation in CHO cells and
discuss its significance for shaping the cell's phenotype. We also look into current state-of-the-art technology that
can be applied to harness and manipulate the epigenetic network so as to nudge CHO cells towards a specific
phenotype. Here, we revise current strategies on site-directed integration and random as well as targeted epi
genome modifications. Finally, we address open questions that need to be investigated to exploit the full
repertoire of fine-tuned control of multiplexed gene expression using epigenetic and systems biology tools.
1. Phenotypic diversity in mammalian production cell lines and especially for human viruses (Stolfa et al., 2018; Frye et al., 2016; Wurm
its ups and downs and Wurm, 2017; Kim et al., 2012; Birch and Racher, 2006). Thus, risks
of immunogenic reactions (due to e.g. differences in protein glycosyla
Chinese hamster ovary (CHO) cell lines are prevalently used for tion) or animal- or virus-derived contaminants are mitigated. Moreover,
biopharmaceutical production of therapeutics (Walsh, 2018) for good the use of CHO cells for industrial production has been optimized into a
reasons: CHO are able to produce proteins with human-like post-trans platform technology over the last 30 years which achieves gram-per-
lational modifications (PTMs), which can be safely administered to pa liter-yields of therapeutic antibodies and other products (Walsh, 2018;
tients, they are easily adaptable to a variety of culture conditions and Berting et al., 2010; Birch and Racher, 2006). Additionally, genetic
medium formulations, enabling growth in serum-free and protein-free engineering of CHO is well established and various technological plat
chemically defined media and they exhibit low virus susceptibility, forms exist to introduce transgenes into its genome.
Abbreviations: 5- Azacytidine, 5-Aza; 5-aza-2′ -deoxycytidine, DAC; 5’-Cytosine-phosphate-Guanine-3′ , CpG; α-1,6-Fucosyltransferase, FUT8; β-Galactoside α-2,6-
Sialyltransferase 1, ST6GAL1; Catalytic domain, CD; cell line development, CLD; Chromatin Immunoprecipitation, ChIP; Chinese hamster ovary, CHO; CRISPR
activation, CRISPRa; CRISPR interference, CRISPRi; Catalytically dead CRISPR associated protein 9, dCas9; Dihydrofolate reductase, DHFR; Double strand breaks,
DSB; gene-of-interest, GOI; Histone deacetylases, HDACs; Homology Directed Repair, HDR; Krüppel Associated Box, KRAB; micro RNA, miRNA; Methotrexate, MTX;
Non-Homologous-End-Joining, NHEJ; Nuclear factor NF-kappa-B p65 subunit, p65; Polycomb group complex, PcG; post-translational modifications, PTM; R
transactivator domain of the Epstein-Barr virus, Rta; Recombinant specific protein productivity, qP; Scaffold/matrix attached regions, S/MARs; Short chain fatty
acids, SCFAs; Single chain variable fragment, scFv; Small interfering RNA, siRNA; Supernova Tagging, SunTag; Translocation methylcytosine dioxygenase 1, TET1;
Ubiquitous chromatin opening elements, UCOEs; Whole Genome Bisulfite Sequencing, WGBS; Yin Yang 1, YY1.
* Corresponding author at: University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
E-mail address: nicole.borth@boku.ac.at (N. Borth).
https://doi.org/10.1016/j.biotechadv.2022.107924
Received 28 December 2021; Received in revised form 3 February 2022; Accepted 4 February 2022
Available online 9 February 2022
0734-9750/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
N. Marx et al. Biotechnology Advances 56 (2022) 107924
Despite the fact that CHO cells are the basis of a multi-billion dollar 2. Phenotypic variability, its sources and consequences
industry (Walsh, 2018, 2014; Birch and Racher, 2006), several funda
mental challenges still exist. A number of publications have reported Potentially, CHO cells gained many of their advantageous charac
production instabilities and unexpected deviation from normal process teristics, such as the ability to grow in suspension and serum-free me
outcome, which could be highly problematic for business and more dium or the ability to produce human-like posttranslational
importantly - in case of immunogenic reactions or supply problems - for modifications, through their adaptability (Wurm and Wurm, 2017;
patients (Osterlehner et al., 2011; Paredes et al., 2013; Kim et al., 2011; Lewis et al., 2013). Cells with desirable phenotypes, e.g. productivity or
Yang et al., 2018; Hossler et al., 2009). Consequently, precise control of growth, that emerge in a cell pool, can be isolated and used to enhance
the cellular phenotype is of utmost importance. For this reason, the biopharmaceutical production processes (Ritter et al., 2016; Bort et al.,
Quality by Design (QbD) concept has evolved which defines acceptable 2010; Pichler et al., 2011; Yamano et al., 2016). The elevated plasticity,
margins for process runs to ensure reproducible product quality and however, is a conflicting feature of CHO cells: while it enables rather
efficacy (US Food and Drug Administration, 2004). simple genome engineering and transgene expression, a phenotypic drift
A plethora of studies have investigated CHO cell lines as the is apparent in long-term cultures. Also, after subcloning inter- and
“workhorse of biopharmaceutical production” to ultimately control and intraclonal differences in phenotypes become apparent, as supported by
enhance cell performance towards better titer, quality, efficacy, process many studies(e.g. (Baik and Lee, 2017; Vcelar et al., 2018b; Patel et al.,
predictability and safety of CHO derived products (Fischer et al., 2015a; 2018; Tharmalingam et al., 2018; Chen et al., 2017; Pilbrough et al.,
Sommeregger et al., 2017; Nagashima et al., 2013; Noack et al., 2015; 2009; Ko et al., 2018; Li et al., 2015)). Various sources for the variability
Hackl and Borth, 2014; Budge et al., 2020; Eisenhut et al., 2020; Amann in behavior between clones and cell lines have been described and
et al., 2019; Yang et al., 2015; Kol et al., 2020; Schmidt et al., 2015; identified in CHO cell research (Fig. 1), mainly focused, for obvious
Tigges and Fussenegger, 2006; Cost et al., 2010; Johari et al., 2015; reasons, on stability of productivity. However, other types of instability,
Fischer et al., 2017). While traditional engineering attempts rather e.g. in growth behavior or product quality, are conceivable and of
focused on media and process optimization (Ritacco et al., 2018; Li relevance.
et al., 2010), new tools that allow dedicated control of (multiple)
endogenous genes or transgene expression have only recently been 2.1. Variation in genome sequence
acknowledged and utilized in CHO cell research (to name a few: Kil
degaard et al., 2013; Brown et al., 2014; Fischer et al., 2015a; Lee et al., Current CHO cell lines are derived from primary fibroblast-like cell
2015; Grav et al., 2015; Bydlinski et al., 2018; Marx et al., 2018; cultures of the Chinese hamster (Cricetulus griseus) and were first isolated
Schmieder et al., 2018; Eisenhut et al. 2018 and 2020; Amann et al., in 1957 (Puck, 1957; Puck, 1986). Since then, different CHO cell lines
2019; Sergeeva et al., 2020; Kol et al., 2020; Karottki et al., 2021; Marx have undergone various types of selection including media adaptation,
et al., 2021). Apart from the feasibility of cell line engineering ap mutations by exposure to gamma rays and ethyl methane sulfonate or
proaches for optimization, a major driver for the use of CHO in bio clonal isolation (Lewis et al., 2013; Wurm, 2013; Wurm and Wurm,
pharmaceutical production has always been its flexibility and 2021). Currently, CHO-K1, CHO-DuxB11, CHO-S and CHO-DG44 cell
adaptability. Established cell line development protocols include the lines and sub-lineages are predominantly used (Lewis et al., 2013; Wurm
screening of thousands of clones and typically result in the isolation of and Wurm, 2017). Compared to the diploid genome of C. griseus, current
multiple clones that yield economically acceptable titers in the gram per cell lines exhibit large variation in chromosome number, size and
liter range. Here, a major concern is the fact that subclones derived from structure (Vcelar et al., 2018b; Vcelar et al., 2018a; M. J. Wurm and
the same, often already subcloned parent, show high diversity with Wurm, 2021; F. Wurm and Wurm, 2017; Derouazi et al., 2006).
respect to the process relevant parameters growth, final cell density and Recently, whole genome sequencing revealed substantial genetic mu
titer. While the diversity in subclone behavior in part is the basis for the tations between CHO cells and the C. griseus genome as well as between
efficiency of cell line development (CLD) protocols, it also is a problem different CHO cell lines and even in subclone populations with impli
as the observed diversity may well include variation in process outcome cations for biopharmaceutical production (Lewis et al., 2013; Brinkrolf
or in quality relevant parameters such as glycosylation, product aggre et al., 2013; Xu et al., 2011; Rupp et al., 2018; Frye et al., 2016;
gation or others (Baik and Lee, 2017; Chen et al., 2017; Ko et al., 2018; Feichtinger et al., 2016; Scarcelli et al., 2018; Kelly et al., 2017; Dhiman
Pilbrough et al., 2009; Strutzenberger et al., 1999; Wurm and Wurm, et al., 2019).
2017; Wurm and Wurm, 2021; Davies et al., 2013). Consequently, FDA Unsurprisingly, high mutation rates can lead to increased cellular
and EMA have established a requirement for documentation of mono diversification in a cell pool and drive clonal variation as observed in
clonality (Welch and Sarah Arden, 2019; US Food and Drug Adminis CHO cells (Wurm and Wurm, 2017; Lewis et al., 2013; Frye et al., 2016).
tration, 1997; International Conference for Harmonisation of Technical Still, small mutations in coding sequences do not necessarily entail a
Requirements for Pharmaceuticals for Human Use, 1997; World Health variation of protein sequences or even the total loss of gene function,
Organization, 2013). This has wide ranging consequences for the in and the vast majority of mutations are located outside coding or even
dustry (Frye et al., 2016), as subcloning is time consuming, elaborate transcribed genes. Although the application of recombinant gene
and thus increases development time and cost. With all other steps in amplification agents can lead to cellular diversification and chromo
CLD pipelines optimized and automated, the time required for a single somal rearrangements (Kim and Lee, 1999; Pallavicini et al., 1990; Kim
cell to expand to sufficient numbers for further testing and processing is et al., 1998; Vishwanathan et al., 2014; Chusainow et al., 2009), it ap
by now the insurmountable limitation for further reduction of time to pears that aberrations arise spontaneously and continuously, as the se
clinic. Thus, undesired and uncontrolled phenotypic variation still rep lection of single cell populations via subcloning is not stabilizing the
resents a major challenge for mammalian cell based recombinant pro genotype nor the chromosome count (Vcelar et al., 2018a, 2018b).
tein production. While the genome of CHO cells is remarkably complete in view of these
In this review we will look into the sources of subclone and pheno chromosomal aberrations, with few regions actually missing compared
type diversity, including unexpected divergence from routine process to the Chinese hamster (Brinkrolf et al., 2013; Lewis et al., 2013), the
performance, with a focus on epigenetic mechanisms that underly this described rearrangements may well lead to changes in gene copy
variation. Furthermore, we provide an overview on current methods and numbers of specific genes, where some genes may become amplified and
technologies that provide the chance to control cellular phenotypes via for others only a single copy remains (Kaas et al., 2015). These copy
epigenetic mechanisms. number variations can have consequences on the gene expression level
of the corresponding genes and thus on the transcriptome and subse
quently on the cellular phenotype. Unfortunately, our understanding of
2
Fig. 1. Main types of instability (A) and possible effect on phenotype (B) in CHO cells.
the effect of mutations in untranslated regions is still very rudimentary – CHO cells have demonstrated that under each given condition, the
questions such as how do point mutations affect promoter or enhancer transcriptomes of individual cells in a given population are remarkably
activity still have no systematic understanding. What is known from uniform, but that the entire population may shift behavior over time (;
cancer research, however, is that such translocations as are frequently Tzani et al., 2021; Ogata et al., 2021).
observed in CHO cells may have a significant impact on gene expression The parameter of phenotypic stability that has received the most
(Harewood and Fraser, 2014; Wang et al., 2020). attention so far, is production stability which is a prerequisite for bio
pharmaceutical manufacturing (it takes weeks to months from thawing
cells to harvesting the desired recombinant protein). Possible causes of a
2.2. Variation of transcriptomes loss of specific recombinant protein productivity during long-term
cultivation include copy number loss as well as gene silencing
An in-house comparison of 150 transcriptomes of cells of various (Table 1). Transgene copy loss (He et al., 2012; Kim et al., 2011) may be
provenience and grown under a variety of culture conditions revealed directly linked to chromosomal rearrangements as well as to the specific
that more than 80% of all expressed genes, in particular those expressed way the transgene plasmid and copies thereof have integrated into the
at higher level, are expressed in all cell lines (unpublished data). Thus, genome, where a high number of repeats may enhance the likelihood of
despite the high variation in genome sequence and chromosome rearrangements and thus copy loss (Dhiman et al., 2020). CHO protein
numbers, the coding transcriptomes of CHO cell lines are remarkably production instability has been investigated thoroughly on various
uniform with respect to which genes are expressed. However, the indi -omics levels over the last decades (Stolfa et al., 2018; Barnes et al.,
vidual gene expression level of each of these thousands of genes are 2003; Kildegaard et al., 2013), including epigenetic changes on the CMV
highly heterogenous providing cells with a wide range of possible promoter that drives productivity. However, the impact of epigenomics
combinations. Therefore, a significant part of the phenotypic variation as a more general, major contributor to differential gene expression on a
observed in CHO cells may be caused not by different genes being global scale, which thus determines phenotypic behavior, has only
expressed or by mutations within the sequence of these genes, but by the recently been addressed in CHO cell research (Feichtinger et al., 2016,
precise combination of gene expression levels across these active genes. Hernandez et al., 2019).
In view of this, the transcriptome of a cell line or culture state provides a
good estimate of this global phenotypic variation, whether it is the
consequence of genome and/or chromosome variation, or of other reg
ulatory mechanisms such as epigenetics. An important aspect in this
context is also the impact of culture conditions and media composition
on cell behavior. Recent publications of single-cell transcriptomes of
3
Table 1 Fisher, 2013; Wu and Sun, 2006; Moris et al., 2016). However, the
Publications reporting variability of protein productivity in CHO cells and the epigenome does not only change due to intrinsic cell signaling, but is
stated different types and sources of instability. also responsive to environmental signals (Feil and Fraga, 2012; Jaenisch
Type of Cause Gene References and Bird, 2003). Aberrant epigenetic regulation is involved in the
instability Amplification development of chronic diseases (Moosavi and Ardekani, 2016) and
(Ritter et al., 2016; Ko et al., prevalent in certain types of cancer (Kulis and Esteller, 2010; Cohen
yes 2018; Li et al., 2016; He et al., et al., 2011; Santos-Rosa and Caldas, 2005; Dawson, 2017).
2012)
chromosomal
(Baik and Lee, 2017;
instability 3.2. Epigenetics in CHO cells
no Jamnikar et al. 2015;
Bandyopadhyay et al. 2019)
not stated (Du et al. 2013) Early studies of production instability in CHO cells reported trans
Gene copy (Morrison, McMaster, and gene silencing by epigenetic mechanisms, which resulted in reduced
loss Piret 1997; S. J. Kim et al.,
specific productivity in CHO cells (Table 1), highlighting the impact of
1998; Fann et al. 2000; M.
yes
Kim et al., 2011; Osterlehner
gene (dys)regulation as a major cause of gene expression instability.
not stated et al., 2011; Beckmann et al. Although these studies were not systematically investigating the cause-
2012) effect relationship between epigenetic marks and gene expression, the
(Yoshikawa et al. 2000; N. S. results complement the genetic sources of instability and might explain
no Kim et al., 1998; J. H. M. Yeo
the frequently observed differential gene expression over time and
et al. 2017)
(M. Kim et al., 2011; within a cultivation process (Motheramgari et al., 2020; Doolan et al.,
Osterlehner et al., 2011; 2013; Vito and Mark Smales, 2018; Kantardjieff et al., 2010; Vishwa
yes
promoter Moritz, Becker, and Göpfert nathan et al., 2014). A changed epigenome is also a common charac
methylation 2015)
teristic of immortalized cells (Futscher, 2013; Novak et al., 2009; Liu
(Patel et al., 2018; Y. Yang
no
et al., 2010)
et al., 2005), harbouring both hypo- and hypermethylated regions in
Gene (Moritz et al. 2016; Veith their genome (Kulis and Esteller, 2010) as well as varying histone
histone yes
silencing
modification
et al., 2016) modifications (Cohen et al., 2011; Santos-Rosa and Caldas, 2005).
no (Paredes et al., 2013) Initially, epigenetic studies in CHO cells focused on the effect of locus
(Strutzenberger et al., 1999;
specific transgene expression. In particular, several research groups
yes Chusainow et al., 2009; H. Li
not stated et al., 2015) could show that DNA methylation or histone modifications deposited at
(J. H. M. Yeo et al. 2017; transgene promoters correlate with recombinant protein production
no
Jamnikar et al. 2015) instability (Table 1, also Wippermann and Noll, 2017). While these
(Strutzenberger et al., 1999; findings demonstrated the importance of epigenetics for phenotypic
Pilbrough et al., 2009; Böhm
et al. 2004; Bailey et al. 2012;
characterization and perception of CHO cells, a deeper understanding of
Subramanian et al. 2018; the general mechanism of epigenetic expression regulation was still
Derouazi et al., 2006; Dorai lacking. In 2015, Wippermann et al. (2015) demonstrated that repres
yes
et al. 2012; Kaneko, Sato, and sion by DNA methylation is locus-dependent in a CHO DP-12 cell line.
Not stated
Aoyagi 2010; Voronina et al.
or N/A Interestingly, cancer cell lines resemble the here observed characteristic
2016; Orellana et al. 2015;
unknown
Tzani et al., 2021; Lee et al., closely, which in turn is in accordance with the cancerous phenotype
2021) inherent in CHO cells. In a comprehensive study that made the complete
(Strutzenberger et al., 1999; data publicly available, Feichtinger et al. (2016) combined global DNA
no Böhm et al. 2004; Pichler methylation, histone modification and genome modification data of
et al., 2011)
not stated (F. Chen et al. 2012)
different CHO cell lines that were either subjected to different evolu
tionary pressures or long-term cultivation in order to record and analyze
the effect of epigenomic and genomic modifications on the CHO
3. Epigenetic regulation of gene expression phenotype. Interestingly, the status of DNA methylation in CHO cells
was largely unchanged when cells are cultured over a prolonged period
3.1. Mechanisms of epigenetic control under constant culture conditions, which underlines the assumption that
DNA methylation functions as a means to transmit a transcriptomic
In mammalian cells, DNA and genetic information is packaged into expression pattern to progeny cells. A change in culture conditions on
chromatin and gene expression can be regulated depending on the the other hand, e.g. the transition from adherent to suspension cells or
chromatin conformation (Fig. 2) (Kouzarides, 2007; Felsenfeld et al., the absence of an important energy substrate (here: L-glutamine),
1996). The modulation of chromatin structure is governed by epigenetic resulted in a strikingly different methylome and highlights epigenetic
mechanisms that can fixate inheritable gene expression patterns without transcriptional regulation as a response to evolutionary pressure. In
changing the underlying DNA sequence (Allis and Jenuwein, 2016). 2019, Dhiman et al. were able to demonstrate the impact of DNA
These epigenetic changes entail post-synthetic modifications of DNA methylation on the energy metabolism of CHO cells that resulted in
nucleotides or associated histone proteins (See Box 1), as well as signatures that characterized low or high producing cell lines (Dhiman
nucleosome remodeling and substitution of histone variants, which, et al., 2019).
amongst others, inflect the topology of the chromatin landscape, alter In contrast to DNA methylation, histone modifications were shown to
the accessibility of transcription factor binding sites or recruit proteins be dynamically modulated in a batch culture, suggesting that short-term
that enhance or repress gene transcription (Cohen et al., 2011; Santos- adaption due to e.g. nutrient limitation is executed via this more rapidly
Rosa and Caldas, 2005; Attwood et al., 2002; Bird, 2002; Cedar and responsive regulatory control system (Hernandez et al., 2019). Addi
Bergman, 2009; Jurkowska and Jeltsch, 2016; Thurman et al., 2012; tionally, a set of histone modifications as well as CpG DNA methylation
Clapier et al., 2017; Deaton and Bird, 2011). Adaptation of these mod was mapped to either gene regulatory elements, active transcriptional
ifications are essential during development and cell differentiation sites or repressed heterochromatin by comparing and annotating Chro
processes, where specific genes are either activated or repressed in a matin Immunoprecipitation (ChIP)-Sequencing and Whole Genome
time-dependent manner that in turn determine cell-fate (Kiefer, 2007; Bisulfite Sequencing (WGBS) data (Fig. 3) (Feichtinger et al., 2016).
Cheedipudi et al., 2014; Atlasi and Stunnenberg, 2017; Cantone and Prominently, the DNA of active promoter regions marked with strong
4
Fig. 2. Illustration of nucleosome organization and epigenetic mechanisms that govern gene expression. Chromatin fibers are compacted nucleosomes that consist of
histone proteins and DNA. Repressed heterochromatin and transcriptionally permissive euchromatin are marked by distinct epigenetic signatures. The best studied
histone modifications in CHO cells and 5’-Cytosine-phosphate-Guanine-3′ (CpG) DNA methylation are depicted.
enrichment of H3K27ac (chromatin state 9) were hypomethylated for in the past (e.g. the set of expressed genes) and aims to rapidly modulate
expressed genes and fully methylated for silenced genes (active pro their expression to find a pattern that suits the new culture conditions or
moter histone marks were depleted simultaneously), verifying the ON/ is able to overcome the specific stress experienced.
OFF function of promoter methylation and highlighting the interplay Intriguingly, another recent study demonstrated that a similar
between DNA methylation and histone modifications in CHO cells. cellular phenotype, in this case improved growth characteristics of three
A recent study investigating the epigenetic impact of subcloning distinct CHO host cell lines, does not necessarily build on similar gene
showed that a new and distinct DNA methylation pattern emerged expression patterns, but does bear resemblance in the regulated path
during the subcloning process in each subclone (Weinguny et al., 2021), ways (Weinguny et al., 2020b). These findings suggest that due to their
which could explain the phenotypic diversity observed between cell innate complexity, mammalian cells can find various routes to achieve
clones, even if they are derived from an already subcloned parent (Pil similar endpoints. Combined, these two studies implicate that, upon
brough et al., 2009; Strutzenberger et al., 1999; Chen et al., 2017; being exposed to environmentally problematic situations (in this
Weinguny et al., 2020b; Tharmalingam et al., 2018; Ko et al., 2018). The particular example the subcloning process of CHO cells), the diversifi
study of Weinguny et al. (2021) demonstrated that differentially meth cation of the transcriptome of mammalian cells is induced by epigenetic
ylated regions were enriched around transcription start sites and/or in alterations, which ultimately allows some of the cells to overcome the
regulatory and enhancer regions, further underlining the hypothesis of specific challenge. Whether the epigenetic response to stress factors is
epigenetics-driven control of the transcriptome variety in CHO cells. targeted (downregulation of genes that are gratuitous) or completely
Interestingly, actively transcribed regions of the genome were exempt random (survival of the fittest) remains to be elucidated. This strategy is
from DNA methylation changes, while promoter regions were affected more likely to produce suitable results than to “hope” for a supportive
only to the extent explained by a completely random distribution across mutation or rather, a combination of mutations to be present in the
the entire genome. While the methylation status of promoters often population, which can then be selected for. An additional evolutionary
determines whether a gene is expressed or not expressed, enhancer advantage is that once a suitable DNA methylation profile has been
functions are more dynamic and can regulate the expression levels of found, this will be passed on to progeny cells to provide them with a start
gene(s). Consequently, the observed changes in DNA methylation pre advantage for further growth. Recently, the comparison of two different
dominantly in regions that exert control over expression level (e.g. en host cell lines showed that the DNA methylation pattern as well as the
hancers) rather than in regions that turn gene expression on or off (e.g. expression level of key epigenetic enzymes of superior production sub
promoters) and the complete absence of any changes in transcribed re clones was inherited from the host cell lines and suggests that selection
gions which need to be fully methylated, indicates that the predominant of preferable cell lines can be already made at very early stages of the
response to any stress or pressure is an attempt by the cells to fine-tune CLD process based on epigenetic patterns (Chang et al., 2022).
the expression of the given, already expressed set of genes, rather than Another important regulatory mechanism was found to be mediated
the activation of new genes or the silencing of an already expressed by lncRNAs, which constitute a major part of ncRNAs in mammalian
gene. In essence, the cell builds on a system that has already worked well genomes (Kashi et al., 2016). lncRNAs were identified that are
5
Box 1
DNA methylation
DNA methylation involves the addition of methyl groups to cytosine or adenine to form 5-methylcytosine (5mC) or 6-methyladenine (6mA),
respectively. The former of these modifications normally occurs in a 5'-cytosine-phosphate-guanine-3’ (CpG) context, although non-CpG
methylation has been reported, but to a much lesser extent [113,114]. Very recently, 6mA modification has been described in eukaryotes
[115–117] and mammalian tissue [118] and has been shown to be involved in epigenetic silencing [119]. However, little is known about its
exact role in epigenetic regulation.
DNA CpG hypermethylation of promoters has been associated with repression of the associated gene, while gene-body methylation is linked to
active transcription [18,99,120,121]. Altogether, DNA methylation is considered to be stably inherited during replication [125,126] and to
serve as a long-term memory for gene silencing in somatic cells [127,128]. In mammals, DNA methylation and demethylation are established by
DNA methyltransferases (DNMT) and Ten-Eleven-Translocation dioxygenases (TET demethylases).
Histone modifications
In eukaryotes, DNA together with histone proteins form a chromatin complex, the nucleosome. As N-terminal histone tails protrude from the
minor groove of the DNA, post-translational modifications of core histone tails can influence accessibility and condensation of chromatin and
thus gene transcription [69,154–156]. A multitude of PTMs were hitherto identified and include i.a. acetylation, methylation, phosphorylation,
ubiquitination, sumoylation, biotinylation, glycosylation, hydroxylation, malonylation and several others [154,157]. Exact function and
interaction partners of many modifications, however, remain largely unknown. Primarily, histone tail modifications recruit different sets of
effector proteins that either activate or repress gene transcription directly or indirectly [154]. Thus, changes in either the PTMs of histone tails,
perturbations of the writers and erasers thereof or the modulation of effector proteins can have tremendous impact on gene expression.
Non-coding RNAs
Non-coding RNAs (ncRNAs) cover a wide range of transcribed but untranslated RNA molecules, of which some play crucial roles in epigenetic
programming. Of these, long non-coding RNAs (lncRNAs) and small non-coding RNAs such as short interfering RNAs (siRNAs) and micro RNAs
(miRNA) have been most frequently studied in CHO cells. These two families of non-coding RNAs are classified by length, where lncRNAs exceed
200 nucleotides. lncRNAs have been correlated to gene regulation and differential gene expression in CHO cells. Best studied is their role in the
process of X-chromosome inactivation. While maintained by DNA methylation, the lncRNA X-ist initiates the repression by binding to the second
X-chromosome and excluding the transcription machinery and recruiting the polycomb group complex (PcG), which mediates gene repression
[161,162]. Other lncRNAs, e.g. HOTAIR, have been described to drive histone modification with a wide targets range [163].
Infobox Figure: Possible interplay between histone modifications, DNA methylation and lncRNAs. Writers, readers and erasers (WRE)
govern the maintenance and modulation of epigenetic marks.
Interaction between different epigenetic marks
Different epigenetic effectors can play crucial roles in gene regulation in mammalian cells. Most of these events are highly interconnected and
include a set of epigenetic modifications to exert the regulatory effect. Extensive research has been conducted in the field of embryonic
development to understand the crosstalk between histone modifications to DNA methylation and valuable information has been gathered on
how these fundamental epigenetic regulatory mechanisms shape the transcriptional landscape. For example, the Xist lncRNA recruits PcG
complexes that in turn propagate DNMT-mediated DNA methylation and long-term silencing. Interaction of DNMTs with enzymes of the PcG
group have also been reported in other studies and show that DNA methylation and H3K27me3 modifications lead to persistent gene repression
[173–175]. Moreover, tri-methylation of H3K9 is associated with DNA methylation at promoter sites and loss of either mark can result in
chromatin de-repression [176,177]. It was also shown that DNA methylation inhibits H3K4me3, an active promoter mark [178]. On the other
hand, Methyl-CpG-binding proteins (e.g MeCP2) have been identified that bind to methylated promoters and recruit protein complexes
including HDACs, which results in silencing of the associated gene [94,179]. However, the exact interplay, pattern and deposition for many
specific epigenetic modulations still remains elusive and a large part of epigenetic modifications have not yet been studied in sufficient detail
while the list of new modifications is steadily extended.
6
Fig. 3. Chromatin states of a CHO-K1 cell line adapted to growth in suspension. A) Based on six histone marks (H3K27ac, H3K4me3, H3K4me1, H3K36me3,
H3K9me3 and H3K27me3), eleven chromatin states (left) were analyzed by ChIP-Seq. B) Enrichment of different chromatin states at different regulatory genomic
regions. C). Percent CpG methylation at the chromatin states in the exponential growth phase of a batch culture. Figure adapted from (Feichtinger et al., 2016).
differentially expressed under different culture conditions or were regulatory control (Hernandez et al., 2019).
correlated with growth or productivity of different clones, thus
demonstrating their potential role in epigenetic control in CHO cells 4. Controlling and manipulating the Epigenome
(Vito and Mark Smales, 2018; Vito et al., 2019; Motheramgari et al.,
2020; Novak et al., 2009). A possible mechanism of lncRNA action was The recently gained insight into transcriptional epigenetic regulation
shown to be the interaction with regulatory regions of coding genes, in CHO cells opens up new possibilities for CHO cell research to un
resulting in their correlated expression possibly due to lncRNA executed derstand previously unexplained cellular behavior and allows the
Fig. 4. Schematic illustration of strategies to impact CHO cell production phenotypes. Overview of cell engineering strategies towards stable clones and enhanced
protein productivity. Left: Methods to protect transgene expression from genomic and epigenetic influences by genetic engineering. Right: Either random or targeted
epigenetic modulation can be used to control or fine-tune gene expression levels and thus achieve a desired production phenotype.
7
development and implementation of new engineering strategies for of successful applications of these elements is extensive, starting already
better control of CHO processes. While early studies correlated pro in the 1980's-90's (Klehr et al., 1991; Bode and Maass, 1988). Here, the
duction instability to DNA methylation of the transgene promoter, reader is referred to other, recent reviews that provide in depth infor
which enabled screening methods to discard clones showing these un mation about the functionality and application of the different elements
desirable traits, larger -omics studies observed how chromosomal and in biopharmaceutical applications (Neville et al., 2017; Harraghy et al.,
epigenetic instabilities can influence the general gene expression pattern 2015; Romanova and Noll, 2017).
in a cell, ultimately leading to performance changes of a production cell
line (Li et al., 2015; Feichtinger et al., 2016). However, most of the 4.2. Targeted GOI integration into favorable chromatin regions
studies focus on correlations between epigenetic marks and gene
expression, but do not show a causative relationship. As epigenetic In contrast to specifically guarding the GOI from silencing or
factors seem to play a major and hitherto underappreciated role in modulating its chromatin context, targeting the integration of the GOI
determining CHO cell phenotypes, a better understanding of the role of into favorable chromatin regions is an alternative strategy to circumvent
individual epigenetic marks in gene regulation is needed to resolve the production instability in CHO cells. Targeted integration by CRISPR-
mere correlative, informative value of former studies. Certainly, the true Cas9 relies on the cellular response to DNA double strand breaks
value of understanding the epigenetic impact on the regulatory network (DSB). After the occurrence of DSBs, either Non-Homologous-End-
is the opportunity to leverage the gained knowledge for engineering Joining (NHEJ) (Davis and Chen, 2013; Rodgers and McVey, 2016) or
approaches in order to overcome existing hurdles in mammalian cell Homology Directed Repair (HDR) (Jasin and Rothstein, 2013) are
protein production. Consequently, tools and methods are required to initiated to repair the damaged DNA. By specific GOI vector and gRNA
first and foremost test the effect of specific epigenetic marks in CHO cells design either NHEJ or HDR can be selectively harnessed to integrate the
and investigate the cause-effect relationship on gene expression to ul desired transgenic DNA at a defined genomic locus (Ran et al., 2013;
timately reduce, prevent or counteract process instabilities or even to Sander and Keith Joung, 2014; Sergeeva et al., 2020; Lee et al., 2015).
enhance protein production. Here, the chromosomal structure and the neighborhood of the GOI
Different approaches to alter epigenetic patterns in CHO cells have integration site can have a tremendous impact on the stability of the
been conducted to stabilize or modulate gene expression, initially production cell line, also known as position effects (West and Fraser,
focusing on stabilization of transgene expression, but more recently 2005; S. Li et al., 2016). Thus, identifying regions with high transcrip
expanding into global cellular gene expression patterns using both tional activity, accessible chromatin and low nucleosome occupancy
global and targeted epigenetic editing (Fig. 4). While the application of should allow a strategy for systematic engineering of stable and high-
new strategies in vector design and locus-dependent gene integration is producing CHO cell lines by integrating the GOI in the desired
used as a means to protect from negative epigenetic regulation or to genomic location resulting in an improved, more stable production
avoid regions of chromosomal instabilities, treatment of cells with phenotype (Dhiman et al., 2020; Balasubramanian et al., 2016; Matasci
chemicals has a global impact on the epigenetic signature and thus on et al., 2011; O'Brien et al., 2018; Veith et al., 2016; Koduri et al., 2001).
various genes. Recently, novel cell engineering strategies that either By using site directed integration, various producing CHO cell lines were
induce random epigenetic changes by knock-down of epigenetic en generated, many of which were reported to be stable (Qiao et al., 2009;
zymes or that exert precise epigenetic control by rewriting the epige Baumann et al., 2017; Kawabe et al., 2017; O'Brien et al., 2018; Wang
netic code have been developed and applied in CHO cell research (Marx et al., 2017; Grav et al., 2018; Soo and Lee, 2008; Cacciatore et al., 2013;
et al., 2018; Weinguny et al., 2020a; Marx et al., 2021; Schmidt et al., Zhao et al., 2018; Sergeeva et al., 2020). However, GOI expression after
2015; Jia et al., 2018). While random epigenetic editing might prove to targeted integration into loci with high transcriptional activity is still
be useful during cell line development for selection of clones with widely varying (Zhao et al., 2018) and although reported results appear
enhanced phenotypic traits, targeted epigenetic editing allows to pre promising, further investigation and methodical studies will be neces
cisely dissect the interplay of epigenetic modifications and their impact sary to identify the most effective integration sites. Pristovšek and col
on gene expression in CHO. Combined, these approaches unlock new leagues recently characterized three integration sites in CHO cells lines.
possibilities to identify key epigenetic pathways responsible for CHO They established landing pads for gene cassette exchange in those three
variability and may ultimately allow to overcome existing hurdles in cell sites that allow quick introduction of various transgenic construction
engineering. Specifically, if cellular phenotypes are defined predomi and thus screening of various genetic parts. Intriguingly, the authors
nantly by the precise mixture of expression levels of thousands of indi find reduced relatively homogenous expression levels of the site-
vidual genes, and if these expression levels are controlled by epigenetic directedly integrated gene of interest, suggesting that clonal variation
mechanisms, then the ability to manipulate these in a targeted way (in view of transgene expression) can be reduced by targeted integration
enables a new paradigm of fine-tuned control over cell behavior that in defined genomic loci (Pristovšek et al., 2019). Recently, more
both complements and transcends traditional cell engineering favorable chromatin integration sites for transgene expression in CHO
approaches. cells have been predicted by Dhiman et al. after systematically evalu
ating various stable and instable producer cell lines (Dhiman et al.,
4.1. Optimization of transgene vector design 2020). Hilliard and Lee mined the CHO genome for safe integration sites
by epigenome and transcriptome investigations in producer and non-
Various strategies and methods have been developed to prevent, producer cell lines (Hilliard and Lee, 2021). Although these chromatin
circumvent or antagonize the downregulation of cellular production regions have yet to be verified by targeted integration experiments, the
capabilities due to epigenetic or genetic dysregulation. Within these findings enlarge the community's repertoire of potential transgene
approaches, the optimization of transgene vector design offers a integration sites for methodical cell engineering of stable, high pro
straightforward solution by preventing the inserted gene of interest from ducing CHO cell lines. Still, more steps lie ahead to achieve a thorough
being silenced by maintaining an open chromosome structure or understanding of the interplay between epigenetic marks, epigenetic
shielding the gene-of-interest (GOI) from the neighbouring chromatin modifiers and chromatin structure that is necessary to fully leverage the
environment, especially the spreading of heterochromatin (Wang et al., correlation between epigenetics and transcriptional regulation in CHO
2014; Talbert and Henikoff, 2006). Several genetic elements were cells.
identified of which ubiquitous chromatin opening elements (UCOEs),
methylation-free CpG islands flanked by promoters of housekeeping 4.3. Random global epigenetic editing: chemical treatment
genes, and scaffold/matrix attached regions (S/MARs, most likely a
boundary region for heterochromatin) are most frequently used. The list To generally enhance cell line performance, the addition of
8
epigenetic drugs to the cell culture has been widely applied in the CHO can be achieved by traditional genetic engineering approaches and
field. The principal mechanism of the most frequently used compounds, modulating (overexpression, knock-out or knock-down) expression
short chain fatty acids (SCFAs) (e.g. sodium butyrate or valproic acid) levels of key players in the epigenetic network. Early studies showed
and cytosine analogues (i.e. 5-Azacytidine (5-Aza) or 5-Aza-2′ -deoxy improved protein productivity by siRNA and miRNA engineering ap
cytidine (DAC)), is that of globally preventing the deposition of proaches involved in HDAC regulation (Fischer et al., 2015b) or by
repressive epigenetic marks by either inhibiting enzymes involved in knock-out or knock-down of DNMTs (Jia et al., 2018; Schmidt et al.,
such processes or by altering their target sites. 2015). Overexpression of the polycomb group protein Yin Yang 1 (YY1) -
SCFAs are inhibitors of histone deacetylases (HDACs) (Candido et al., a transcription factor that can activate or repress genes depending on its
1978; Davie, 2003; Krämer et al., 2003) and have been employed to binding characteristic to a gene (Gordon et al., 2006, 1; Infante et al.,
enhance production of recombinant proteins since the early 1990's 2015, 1) - showed effects on recombinant protein productivity in CHO
(Palermo et al., 1991; Lamotte et al., 1999; Mimura et al., 2001; Kant cells (Brown et al., 2015; Tastanova et al., 2016). These studies did show
ardjieff et al., 2010; Backliwal et al., 2008). Interestingly, sodium general effects on the CHO transcriptome, although epigenetic analyses
butyrate addition to different mammalian cell lines revealed opposing were not performed at all or were focused on transgene promoters, so
effects dependent on the cell line since the same gene pathways, e.g. that global epigenetic effects cannot be assessed. In a recent study,
apoptosis, were either up- or downregulated upon butyrate treatment Weinguny et al., 2020a performed temporary knock-downs of DNMT1
(Davies et al., 2013; Tabuchi et al., 2002, 2006; Wippermann et al., and DNMT3a as well as TET2 and TET3 enzymes that regulate DNA
2016). Additionally, butyrate treatment has been linked to the Warburg methylation in mammalian cells. Importantly, siRNAs were repeatedly,
effect (Donohoe et al., 2012) - a central metabolic switch in CHO Batch but transiently transfected to reduce expression levels of the respective
and Fed-Batch processes (Mulukutla et al., 2012; Toussaint et al., 2016) - genes temporarily for a total of 16 days in CHO-K1 cells, as a complete
that bears great importance for CHO bioprocesses. knockout of DNMTs is lethal. Contrary to small chemicals, this approach
The nucleoside analogues 5-Aza or DAC are incorporated into the directly targets an epigenetic control mechanism and thus has less side-
DNA during replication and are recognized by the DNA methyl effects. Whole genome bisulfite sequencing revealed efficient methyl
transferase machinery, whereby the enzyme is trapped by a covalent ation pattern alterations in the genome upon KD of DNMTs and to a
bond, which triggers its degradation by the DNA damage signaling lesser extent for TETs. As a result of random changes in the DNA
pathway, thus resulting in global depletion of DNMTs and cytosine methylation pattern of individual cells caused by the downregulation of
methylation during the replication process (Stresemann and Lyko, the enzymes that maintain the existing pattern, the population diversi
2008). Notably, up to 90% of 5-Aza can be incorporated into RNA fied with respect to cell specific productivity, thus enabling the sorting
instead of DNA due to its ribonucleoside nature (Li et al., 1970), of new cell lines with up to 1.5-fold increased titer and qP (Weinguny
therefore also affecting RNA methylation. The incorporation into et al., 2020a). Noteworthy, due to the inherent randomness of this
transfer RNAs in turn inhibits tRNA methyltransferases and thus tRNA approach and the complexity of epigenetic regulation, the introduction
methylation, which impacts ribosomal RNA processing (Lee et al., 1997; of a unidirectional change that provokes a global regulatory imbalance
Weiss and Pitot, 1974; Glazer et al., 1980), so that the exact effector will also create undesirable phenotypic traits in some cells in the pop
function of 5-Aza on protein synthesis has to be attributed to various ulation. This study describes one of the first approaches that employed
modulations. DAC, a deoxyribonucleoside, on the other hand is incor intentional diversification of a CHO cell population based on in
porated into DNA only and might be therefore more potent to aid in DNA terferences with the epigenome to isolate new cell lines with improved
demethylation than 5-Aza (Li et al., 1970). Still, the side-effects of these traits and thus underlines the importance of epigenetic marks on the
chemicals are manifold (e.g. reduced growth, toxicity, chromosomal behavior in mammalian production cell lines.
instability (Yang et al., 2010; Davidson et al., 1992; Matoušová et al., Whilst most studies so far focus on productivity and expression sta
2011)). bility, possible unwanted and unforeseen effects in other pathways (e.g.
Although the addition of chemical reagents to CHO cell cultures to secretory, ubiquitin-proteasome, glycosylation etc.) relevant for the
increase protein productivity or prevent the instability thereof has quality of the biopharmaceutical have not yet been investigated, but
shown that epigenetic mechanisms do influence essential process pa should be included in the future.
rameters and can be modulated for improved output, the lack of
knowledge of how these reagents exactly modulate gene expression and 4.5. Targeted epigenetic editing
their various side effects (Yang et al., 2010; Mimura et al., 2001;
Davidson et al., 1992; Matoušová et al., 2011) hinder the full use of these Targeted epigenetic editing of selected genomic loci can overcome
strategies for systematic cell line engineering. In addition, as most of off-target prone induction of global epigenetic changes and directly link
them have potentially toxic side effects, their applicability in a pro the cause-effect relationships between differential gene expression and
duction process of a therapeutic protein would be limited due to regu specific epigenetic marks. Rewriting the epigenetic code of specific loci
latory concerns. has only recently been adopted as a result of disruptive technologies, i.e.
It is noteworthy, that while the precise mechanism of their interac the availability of more complete CHO and Chinese hamster genomes
tion with the epigenome is not fully understood, chemicals that are the (Brinkrolf et al., 2013; Lewis et al., 2013; Xu et al., 2011; Rupp et al.,
most frequently used ones in traditional gene amplification methods, 2018) and the discovery and implementation of CRISPR-based genome
such as methotrexate, also have an impact on epigenetic patterns (Nair engineering strategies.
et al., 2020; Gosselt et al., 2021). Thus, their traditional use during cell The basic principle behind targeted epigenetic editing is the fusion of
line development may have contributed to the selection of well per catalytic enzyme domains of known epigenetic modulators with cata
forming subclones by enabling cells to modulate their gene expression lytically inactive molecular DNA targeting tools (Kungulovski and
pattern in such a way that they are able to handle the high load of Jeltsch, 2016; Rots and Jeltsch, 2018; Enríquez, 2016). Due to their ease
transgene cargo generated by amplification of the GOI. In fact, Vish of use, low cost and multipurpose application, CRISPR-based tools (i.e.
wanathan et al. (2014) showed just that: after initial increases in gene catalytically dead Cas9 (dCas9) (Qi et al., 2013)) have been the method
copy number, further raises in methotrexate concentration did not result of choice as illustrated by the multitude of publications in this field (to
in more gene copies, but in changes in transcriptome pattern. name a few: Qi et al., 2013; Maeder et al., 2013b, 2013a; Huang et al.,
2017; Choudhury et al., 2016; McDonald et al., 2016; O'Geen et al.,
4.4. Random global epigenetic editing: (epi)genetic engineering 2017; Xu et al., 2016; Marx et al., 2018; Morita et al., 2016; O'Geen
et al., 2019; Stepper et al., 2017, 9; Kleinjan et al., 2017, 9; Vojta et al.,
A more direct approach to reprogram the cell's epigenetic landscape 2016; Konermann et al., 2015; Shen et al., 2017; Chavez et al., 2016; Yeo
9
et al., 2018; Xiong et al., 2019; Marx et al., 2021; Karottki et al., 2020; 2015). Targeted DNA methylation and DNA demethylation have been
Gilbert et al., 2013, 2014; Hilton et al., 2015). By introducing point among the first studies of dCas9-based epigenetic editing (Maeder et al.,
mutations (D10A and H840A) in the two catalytic domains (RuvC and 2013b; Huang et al., 2017; Morita et al., 2016; Vojta et al., 2016; Stepper
HNH) of the Cas9 endonuclease, a targeted dCas9 molecule solely binds et al., 2017; O'Geen et al., 2019; Marx et al., 2018, 2021; Xiong et al.,
to DNA without introducing double strand breaks (Enríquez, 2016; Qi 2019). However, the studies are not well comparable as different targets,
et al., 2013). Thus, by fusion with other proteins the CRISPR/dCas9 cell lines, dCas9 configurations and DNA methylation analyses have
complex can be used to target theoretically any epigenetic effector to been used. Results indicate, however, that using the dCas9-SunTag
specific genomic loci in order to study their direct effect on gene system results in superior editing efficiency and gene expression mod
expression. Initially, epigenetic effector domains were fused to dCas9 ulation (Morita et al., 2016; Pflueger et al., 2018; Lei et al., 2018).
via amino acid linkers of variable length. Linker length plays an In CHO cells, few targeted epigenetic editing studies have been
important role for the efficacy of dCas9-based editing tools, but has to be published so far. Shen et al. (2017) reported a method to enhance
adjusted depending on the effector domain used (Morita et al., 2016; transgene expression by epigenetically repressing the dihydrofolate
Guilinger et al., 2014; Pulecio et al., 2017). Efficacy could be further reductase (dhfr) gene, which was incorporated into an expression vec
improved in some cases by multiplexed gRNA targeting (Maeder et al., tor, with a dCas9-KRAB construct in a DHFR− cell line (Shen et al.,
2013b; McDonald et al., 2016; Hilton et al., 2015; Liu et al., 2016), 2017). In the native state the Krüppel Associated Box (KRAB) protein
amplification of effector occupancy by introduction of binding sites in binds to DNA via its C2H2 zinc finger domain and represses transcrip
the gRNA of the bacteriophage coat protein MS2 (Chavez et al., 2016; tion locally by interacting with other enzymes involved in histone
Konermann et al., 2015), or by applying the Supernova Tagging system methylation (e.g. the histone-lysine N-methyltransferase SETDB1) or
(SunTag), a fused GCN4 peptide array to dCas9 that serves as an antigen histone deacetylation (e.g. the Nucleosome Remodeling Deacetylase
for a single chain variable fragment (scFv) (Fig. 5) (Huang et al., 2017; NURD). The repression of dhfr by CRISPR interference (CRISPRi) while
Morita et al., 2016; Tanenbaum et al., 2014). For epigenetic editing, undergoing MTX selection resulted in 3.6-fold higher GFP copy numbers
effector domains were fused to MS2 or scFv proteins, respectively. and 3.8-fold higher GFP expression, which was used as a model protein
Different epigenetic effector domains have been tested together with to measure productivity, compared to control cultures. In contrast to the
the dCas9 system including transcriptional repressors, activators, his chemical approaches mentioned earlier, no detrimental effect on cell
tone modification enzymes and DNA (de)methylases (Mali et al., 2013; growth was observed. Similarly, dCas9-KRAB was used to downregulate
Kungulovski and Jeltsch, 2016; Rots and Jeltsch, 2018; Enríquez, 2016; apoptotic genes in CHO cells, which resulted in modest increases in
Maeder et al., 2013b; Choudhury et al., 2016; O'Geen et al., 2017; viable cell density 24 h after transfection (Xiong et al., 2019). Kleinjan
Morita et al., 2016; Pulecio et al., 2017; Konermann et al., 2015; Vojta et al. (2017) on the other hand applied a CRISPR activation (CRISPRa)
et al., 2016; Kleinjan et al., 2017; Stepper et al., 2017; Kearns et al., approach to activate endogenous genes (rasL11a and arpc1b) in CHO
Fig. 5. Different dCas9 systems and epigenetic effector domains for transcriptional regulation in mammalian cells. A) From left to right: dCas9 fused via an amino
acid linker to an effector domain. Different repressor or activator domains can be applied to site-specifically control gene expression. The dCas9-SunTag system
consists of a dCas9 protein fused to a GCN4 peptide array that serve as antigens for scFv fragments fused to effector domains. Dependent on the number of GCN4
antigens. The presence of the effector domain is multiplied. Viral coat proteins, e.g. MS2, fused to effector domains can bind to motifs within a modified gRNA
structure. Different coat proteins can be used for simultaneous and multiplexed epigenetic modulation of an individual genomic locus. Integration of lncRNA se
quences into the gRNA can allow sequence-specific and lncRNA-dependent modifications. B) Epigenetic effector that have been used in CHO cells. Left: transient
effects have been reported with KRAB, PR and VPR domains. The resulting epigenetic changes have been reported for other mammalian cell lines. Right: stable
epigenetic editing has been achieved via targeted DNA methylation or demethylation. Histone modifications are autologously adapted by HDACS, HATs, HDMTs and
HMTs after modulation of the DNA methylation status.
10
cells (Kleinjan et al., 2017) by fusing the nuclear factor NF-kappa-B p65 cells. Recently, Cas13 was identified as an RNA-guided RNAse that can
subunit (p65) (Nolan et al., 1991) and the R transactivator domain of the be repurposed for RNA base editing (Cox et al., 2017; Abudayyeh et al.,
Epstein-Barr virus (Rta) (Hardwick et al., 1992) to yield dCas9-PR. 2016, 2017; Shen et al., 2020) and might allow the modulation of RNA
Additionally, an Auxin-Inducible Degron domain was fused to the modifications such as N6-methyladenosine or 5-methylcyosine, which
dCas9 domain so that the fusion protein would be degraded upon the correlate with protein translation (Noack and Calegari, 2018). The
addition of auxin. The authors could demonstrate that the strength of integration of long non-coding RNAs (up to 4.8 kb) into the gRNA
gene upregulation was inversely correlated with auxin concentration. A structure of the dCas9-complex enabled targeted activation or down
CRISPRa system with three activation domains (VP64, p65 and Rta) was regulation of specific loci dependent on the activity of the lncRNA
also used to activate previously silenced glycosyltransferases (Karottki (Shechner et al., 2015), which could be leveraged to further investigate
et al., 2020). However, most of these studies generated only transient and confirm results of recent CHO-specific lncRNA profiling studies
activation or silencing of the target genes. Stable expression of the (Fig. 5) (Hernandez et al., 2019; Vito and Mark Smales, 2018; Mother
epigenetic modulation tools, however, results in overexpression stress amgari et al., 2020).
and draws protein production capacity, thus limiting the application
range. Therefore, the current concepts of CRISPRi and CRISPRa are best 5. Outlook
used for screening purposes that are then replaced by other established
technologies such as overexpression or gene KO. Still, these tools are Using targeted epigenetic editing tools to control gene expression
useful to 1) study the impact of specific chromatin modifications on gene provides an additional promising advantage over traditional cell line
expression levels, 2) modulate expression levels of selected genes in a engineering for improved cell culture performance: commonly, gene
more native range, which can allow to analyze their effect on meta function studies apply either knock-out or stable overexpression of the
bolism and 3) apply multiplexed up- or downregulation strategies to transgene and both techniques alter the genome and genomic sequence.
analyze metabolic pathways. Stable overexpression, on the other hand, requires gene integration,
Stable manipulation of epigenetic expression control indicating a typically at a random position of the genome, where unidentified
direct cause-effect relationship between promoter methylation and gene genomic regions may be disrupted and their function destroyed in situ.
expression was reported by Marx et al. in 2018. By application of dCas9- This might affect both coding genes and regulatory regions with un
based DNA demethylation and methylation, stable activation and sub predictable consequences. Destabilizing the latter may turn out to be
sequent re-silencing of the β-Galactoside α-2,6-Sialyltransferase 1 more crucial because their function is even less studied and understood
(st6gal1) gene in a CHO cell line was achieved. By coupling the catalytic than those of coding genes. For both knock-outs and knock-ins, there
domain (CD) of Ten-Eleven Translocation methylcytosine dioxygenase 1 fore, subcloning is required which again gives rise to subclone variation
(TET1) with dCas9 and targeting this complex to the promoter of the and consequently requires extensive characterization of many clones so
beforehand silent st6gal1 gene, it was activated as shown on a tran as to unambiguously analyze the effect of the studied gene. In contrast,
scriptomic (mRNA) and reaction (N-glycan analysis) level. The succes epigenetic engineering leaves the genomic context unaltered and, as it is
sive re-methylation of the promoter via a complex of DNMT3A-CD and inherently modifiable, can be reversed to also observe the expected
dCas9 resulted in effective downregulation of st6gal1. While growth and reversal of the phenotype. Additionally, the high percentage of modified
recombinant protein productivity were not affected, the single transient cells in a cell pool (up to 60%) allows for rapid analysis without the
transfection with the dCas9-TET1 tool resulted in stable activation of the absolute need for subcloning (Marx et al., 2018).
st6gal1 gene over more than 80 days, which serves as proof-of-concept Although targeted epigenetic editing is a promising technology to
that DNA methylation is a mechanism for long-term regulation of gene elucidate the epigenetic network in cells and to change expression of
expression patterns in CHO cells. The dCas9-SunTag system was endogenous genes in a more native context, current tools are still limited
furthermore used to investigate the consequential effects of DNA in their applicability for cell engineering. Transient modulations by
methylation on chromatin patterns of endogenous and viral promoters CRISPRa/i last only a few days (oftentimes <48 h), thus complicating
(i.e. CMV) (Marx et al., 2021). Enhanced targeted promoter methylation the analysis of their effect on gene pathways that might need more time
by fusing DNMT3A-CD with the N-terminus of DNMT3L resulted in to establish a novel phenotype. Additionally, fine-tuning to an exact
strong and stable downregulation of the previously activated st6gal1 gene expression level cannot be accomplished currently. Similarly, by
and natively expressed α-1,6-Fucosyltransferase (fut8) genes. CMV- targeted methylation or demethylation, although resulting in inherit
driven BFP expression on the other hand, although responding, was able gene expression changes, genes can only be turned on or off, but
less affected by targeted DNA methylation. Importantly, artificial intermediate expression levels cannot be achieved or controlled. On the
changes in DNA methylation lead to autologous adaptation of histone other hand, due to the inherently targeted nature of CRISPR based
modifications at the targeted regions. Histone marks associated with technologies, these tools could easily be applied to modulate multiple
active promoters are depleted by the cell upon DNA methylation and are genes simultaneously, which might be needed to nudge cells towards
added upon DNA demethylation. Accordingly, repressive histone marks improved production phenotypes.
were added upon DNA methylation and removed after DNA demethy Thus, despite the progress made in the last years, epigenetic research
lation by the cell's regulatory system. Interestingly, the data demon for production cell lines is still in its infancy and further hurdles must be
strated that DNA methylation can nudge the specific type of gene overcome to eventually harness this novel regulatory layer for indus
repression into a poised (facultative heterochromatin) or permanent trially relevant applications. It is still unclear whether the cell's epige
(constitutive heterochromatin) state depending on the native state of netic response to environmental changes, such as changing nutrient
expression of the respective gene. This indicates that cells are able to availability (Batch and Fed-Batch processes) or temperature shift (Fed-
respond to additional, yet uncharacterized chromatin state marks. Apart Batch), is following a reproducible pattern or whether phenotypes
from elucidating epigenetic mechanisms that might help to augment evolve randomly. While changes in DNA methylation have been
selection strategies, targeted DNA methylation or demethylation might demonstrated to occur during subcloning, other typical cell culture op
become a useful tool for cell line engineering since i) no stable inte erations that may be stressful still need to be tested for their effect on the
gration of the plasmids is necessary, ii) genes can selectively be turned epigenome: adaptation to different culture media, freeze/thaw cycles,
on or off and iii) the effects are stably inherited to daughter cells. passaging schedules (every 2–3 days vs 3–4 days), scale-up, transfection
Apart from applications of enzyme domains that modulate DNA- stress and many others. It would be highly interesting to analyze
encompassed epigenetic signatures, the CRISPR complex's plasticity al whether a universal mechanism is activated upon different types of
lows for a wider scope of targets in the cell, for instance by modulation of stress or whether different stress types induce different pathways of
mRNA or by targeting functional RNAs to specific target sites in CHO response. Therefore, factors or growth conditions that might minimize
11
unwanted de-regulation and thus enable a more robust and stable pro CRediT authorship contribution statement
cess operation need to be identified. In the context of manufacturing, the
epigenetic response to a changing environment might be especially Nicolas Marx: Writing – original draft, Writing – review & editing,
important for the choice of cell culture process. Batch or Fed-Batch Visualization, Project administration, Conceptualization, Data curation,
cultures display constantly changing levels of nutrients and waste Supervision. Peter Eisenhut: Writing – original draft, Writing – review
products that can lead to unwanted epigenetic responses (Wingens et al., & editing, Visualization, Conceptualization, Data curation. Marcus
2015). Additionally, epigenetic factors have been linked to the Warburg Weinguny: Writing – original draft, Data curation. Gerald Klanert:
effect (Jing et al., 2020; Donohoe et al., 2012; Zhang et al., 2019), the Writing – original draft, Conceptualization, Data curation, Supervision.
mechanism responsible for the uptake of lactate in the stationary phase Nicole Borth: Writing – original draft, Writing – review & editing,
of a standard Fed-Batch process. Perfusion processes, with mostly stable Project administration, Conceptualization, Supervision, Funding
steady state characteristics of media components and cell densities, acquisition.
might be better suited to circumvent such environmentally induced
epigenetic changes. It was recently reported that a CHO cell perfusion Declaration of Competing Interest
process only showed minimal differential gene expression whereas the
transition from a perfusion pre-culture to a Fed-Batch process resulted in Authors hereby confirm that they have no conflict of interest.
substantial differential expression (Stepper et al., 2020). Apart from
rather well-described epigenetic processes, such as DNA methylation, References
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Slaymaker, Ian M., Cox, David B.T., Shmakov, Sergey, et al., 2016. C2c2 Is a single-
nerström et al., 2019), are constantly emerging and provide windows of component programmable RNA-guided RNA-targeting CRISPR effector. Science 353
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How To Train Your Cell - Towards Controlling Phenotypes by Harnessing The Epigenome of Chinese Hamster Ovary Production Cell Lines

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