Enhanced Genome Editing Tools For Multi-Gene
Deletion Knock-Out Approaches Using Paired CRISPR
sgRNAs in CHO Cells
Valerie Schmieder, Nina Bydlinski, Richard Strasser, Martina Baumann,
Helene Faustrup Kildegaard, Vaibhav Jadhav,* and Nicole Borth*
Since the establishment of clustered regularly interspaced short palindromic repeat
(CRISPR)/Cas9, powerful strategies for engineering of CHO cell lines have emerged.
Nevertheless, there is still room to expand the scope of the CRISPR tool box for
further applications to improve CHO cell factories. Here, the authors demonstrate
activity of the alternative CRISPR endonuclease Cpf1 in CHO-K1 for the first time and
that it can be used in parallel to CRISPR/Cas9 without any interference. Both, Cas9
and Cpf1, can be effectively used for multi-gene engineering with a strategy based on
paired single guide RNAs (sgRNAs) for full gene deletions. This strategy also enables
the targeting of regulatory regions, which would not respond to the conventional
frameshift mutations, as shown by removing the
-1,6-Fucosyltransferase 8 (FUT8)
promoter resulting in a functional knock-out. FUT8 also served as model to verify
that deletion efficiency is size-independent (2–150 kb). To test the suitability for multi-
gene approaches in combination with gene deletion, clones harboring triple deletions
-1,4-Galactosyltransferase (B4GALT) isozymes are identified using solely conven-
tional PCR/qPCR. In addition, two bicistronic transcription strategies are implemented
to enable unequivocal pairing of sgRNAs: a CHO-derived tRNA linker that works for
used with Cpf1 due to its RNA processing ability. These strategies broaden the range
of application of CRISPR for novel gene editing approaches in CHO cells and also
enable the efficient realization of a genome-wide deletion library.
Chinese Hamster Ovary (CHO) cells are the most widely used
expression system for the production of complex therapeutic
proteins since their establishment as cell
factories in 1987.
Despite the importance
of CHO cells for industry, it was only
recently that genomic and transcriptomic
data of high quality was acquired.
However, now, that such data sets are
available, the focus of optimization of cell
performance has shifted from the original
approach of optimizing media, cell selec-
tion procedures, and production process
conditions, toward directed cell
In parallel, the development of tools for
targeted cell engineering has made a
tremendous leap forward with the intro-
duction of nucleases derived from bacterial
clustered regularly interspaced short palin-
dromic repeat (CRISPR) immune systems.
Cas9, originally identiﬁed as the effector
endonuclease of Streptococcus pyogenes,
has been proven to be an efﬁcient editing
tool in many mammalian cells using a
single chimeric RNA guide (single guide
RNA À sgRNA).
Cas9 introduces DNA
double strand breaks (DSB), which are
repaired by either homology directed repair
or non-homologous end-joining (NHEJ),
with the latter being the more frequent
mechanism in mammalian cells.
usually results in the insertion or deletion (InDel) of nucleotides
at the targeted site. With CRISPR/Cas9, genes can be disrupted
in a low-cost and time-saving manner, while ensuring high
speciﬁcity for a large panel of targets.
Therefore, it is now
possible to bring cell engineering to the next level: by combining
numerous deletions of protein-coding regions and non-coding
regulatory elements, entire cellular pathways can be either
altered or completely blocked.
Cas9 has already been used quite extensively in CHO cells
and further development of CRISPR based tools is happening at
a rapid pace. Cpf1, a CRISPR class 2 type V enzyme, was recently
introduced as an alternative editing system.
Two Cpf1 variants
derived from Acidaminococcus_sp_BV3L6 (AsCpf1) and Lachno-
spiraceae_bacterium_ND2006 (LbCpf1) were already found to be
active in human cell lines.
Published work based on this novel
system is limited and to our knowledge, its use in CHO cells has
not yet been demonstrated. Similar to Cas9, Cpf1 is an
V. Schmieder, N. Bydlinski, R. Strasser, N. Borth
BOKU University of Natural Resources and Life Sciences
Vienna, Austria email@example.com
V. Schmieder, Dr. M. Baumann, Dr. V. Jadhav
Austrian Center of Industrial Biotechnology
H. F. Kildegaard
The Novo Nordisk Foundation Center for Biosustainability
Technical University of Denmark
Kgs. Lyngby, Denmark
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/biot.201700211.
Genomic Engineering www.biotechnology-journal.com
Biotechnol. J. 2018, 13, 1700211 © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1700211 (1 of 10)