CRISPR/Cas9 gene editing for the creation of an MGAT1-deficient CHO cell line to control HIV-1 vaccine glycosylation

PLoS Biology
Gabriel ByrnePhillip W Berman

Abstract

Over the last decade, multiple broadly neutralizing monoclonal antibodies (bN-mAbs) to the HIV-1 envelope protein (Env) gp120 have been described. Many of these recognize epitopes consisting of both amino acid and glycan residues. Moreover, the glycans required for binding of these bN-mAbs are early intermediates in the N-linked glycosylation pathway. This type of glycosylation substantially alters the mass and net charge of Envs compared to molecules with the same amino acid sequence but possessing mature, complex (sialic acid-containing) carbohydrates. Since cell lines suitable for biopharmaceutical production that limit N-linked glycosylation to mannose-5 (Man5) or earlier intermediates are not readily available, the production of vaccine immunogens displaying these glycan-dependent epitopes has been challenging. Here, we report the development of a stable suspension-adapted Chinese hamster ovary (CHO) cell line that limits glycosylation to Man5 and earlier intermediates. This cell line was created using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing system and contains a mutation that inactivates the gene encoding Mannosyl (Alpha-1,3-)-Glycoprotein Beta-...Continue Reading

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Citations

Nov 27, 2019·Briefings in Functional Genomics·Jinyu SunXiaorong Hu
Jul 30, 2020·Protein Engineering, Design & Selection : PEDS·Sara M O'RourkeNikolaos G Sgourakis
Dec 12, 2020·Metabolic Engineering·Keiji Nishida, Akihiko Kondo
Oct 23, 2020·Nature Reviews. Molecular Cell Biology·Katrine T SchjoldagerHenrik Clausen
Feb 23, 2021·The Journal of Biological Chemistry·Yoshiki NarimatsuHenrik Clausen

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Datasets Mentioned

BETA
MG189369

Methods Mentioned

BETA
glycosylation
protein folding
transfection
PCR
MDS

Software Mentioned

Data Explorer
glycowork bench
Expasy
Prism
MaxCyte STX
IDEXX
BLAST

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