CRISPR/Cas9-Mediated Genetic Engineering of Hybridomas for Creation of Antibodies that Allow for Site-Specific Conjugation

: Covalent conjugation of chemical moieties to antibodies has numerous applications, including antibody-drug conjugates, antibody conjugation for diagnostics, and more. Most nonspecific chemical conjugation methods ligate onto any of a number of sites on the antibody, leading to multiple conjugated species, many of which perturb antibody function. To solve these problems, we used CRISPR/Cas9-edited hybridomas to introduce a Sortase tag (LPXTG) and a Flag tag at the 3' end of the CH3 heavy chain region of a mouse monoclonal antibody. The Flag tag allows easy purification of the antibody, while the LPXTG is then acted on by the bacterial transpeptidase Sortase to site-specifically add on any of a number of chemical moieties that possess a triglycine repeat. This technique thus allows rapid production of an antibody onto which a wide array of chemical moieties can be site-specifically conjugated.