Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool.
Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user-friendly bioinformatics tool, named "CRISPy" for rapid identification of sgRNA target sequences in the CHO-K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off-target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells.
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
Carlotta
C
Ronda
Lasse Ebdrup
LE
Pedersen
Henning Gram
HG
Hansen
Thomas Beuchert
TB
Kallehauge
Michael J
MJ
Betenbaugh
Alex Toftgaard
AT
Nielsen
Helene Faustrup
HF
Kildegaard
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D006224:Cricetinae; D003412:Cricetulus; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005647:Fucosyltransferases / Q000235:genetics*; D055786:Gene Knockout Techniques / Q000379:methods*; D016678:Genome; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D054643:INDEL Mutation; D020407:Internet; D018832:Molecular Chaperones / Q000235:genetics*; D008969:Molecular Sequence Data; D017393:RNA Editing; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism
D018832:Molecular Chaperones; D000094704:RNA, Guide, CRISPR-Cas Systems; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase; D004720:Endonucleases
CRISPR Cas9; CRISPy; Chinese hamster ovary cells; database; genome editing