One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment.

The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9-expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off-target effects were observed from off-target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene-disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild-type CHO-S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.

[1]  Smaroula Dilioglou,et al.  Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide–based retroviral vector , 2004, Nature Biotechnology.

[2]  David P. Kreil,et al.  CHO microRNA engineering is growing up: Recent successes and future challenges☆ , 2013, Biotechnology advances.

[3]  David R. Liu,et al.  High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity , 2013, Nature Biotechnology.

[4]  Jean-Claude Martinou,et al.  Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli , 2002, The Journal of cell biology.

[5]  H. Kim,et al.  A guide to genome engineering with programmable nucleases , 2014, Nature Reviews Genetics.

[6]  D. Sorrell,et al.  Targeted modification of mammalian genomes. , 2005, Biotechnology advances.

[7]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[8]  K. Chayama,et al.  Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system , 2014, Scientific Reports.

[9]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[10]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[11]  Alex Toftgaard Nielsen,et al.  Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool , 2014, Biotechnology and bioengineering.

[12]  Edward J. O'Brien,et al.  Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome , 2013, Nature Biotechnology.

[13]  David A. Scott,et al.  Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity , 2013, Cell.

[14]  Yarden Katz,et al.  Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system , 2013, Cell Research.

[15]  David A. Scott,et al.  Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells , 2014, Nature Biotechnology.

[16]  Rudolf Jaenisch,et al.  One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering , 2013, Cell.

[17]  Jeffrey C. Miller,et al.  Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases , 2010, Biotechnology and bioengineering.

[18]  S. Korsmeyer,et al.  Proapoptotic BAX and BAK: A Requisite Gateway to Mitochondrial Dysfunction and Death , 2001, Science.

[19]  L. Presta,et al.  Lack of Fucose on Human IgG1 N-Linked Oligosaccharide Improves Binding to Human FcγRIII and Antibody-dependent Cellular Toxicity* , 2002, The Journal of Biological Chemistry.

[20]  Jin-Soo Kim,et al.  Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases , 2014, Genome research.

[21]  Yolanda Santiago,et al.  BAK and BAX deletion using zinc‐finger nucleases yields apoptosis‐resistant CHO cells , 2010, Biotechnology and bioengineering.

[22]  Eric P. Bennett,et al.  High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs , 2014, Nucleic acids research.

[23]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

[24]  Kelvin H. Lee,et al.  The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line , 2011, Nature Biotechnology.

[25]  Charles A. Gersbach,et al.  Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector , 2014, Nucleic acids research.

[26]  T. Noll,et al.  Methods in mammalian cell line engineering: from random mutagenesis to sequence-specific approaches , 2010, Applied Microbiology and Biotechnology.

[27]  Morten H. H. Nørholm,et al.  A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering , 2010, BMC biotechnology.

[28]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.