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Site-Specific Generation of Protein-Protein Conjugates Using Native Amino Acids

preprint
revised on 14.10.2019 and posted on 16.10.2019 by Marco Lobba, Christof Fellmann, Alan Marmelstein, Johnathan Maza, Elijah Kissman, Stephanie A. Robinson, Brett T. Staahl, Cole Urnes, Rachel J. Lew, Jennifer A. Doudna, Matthew Francis, Casey S. Mogilevsky

Chimeric protein-protein conjugates provide platforms for immunotherapy, targeted drug delivery, and vaccine development. However, many desirable constructs cannot be produced through direct expression, and the targeted coupling of two proteins is chemically challenging. Here we present a new approach for the rapid and site-specific coupling of proteins using native amino acids. Tyrosinase oxidizes exposed tyrosine residues on polypeptides, generating ortho-quinones that react rapidly with strategically placed cysteine residues in other proteins. This approach was used to modify CRISPR-Cas9 and other substrates with small molecules, peptides and even intact proteins. The conjugation of cell penetrating peptides to CRISPR-Cas9 was shown to increase cellular genome editing efficiency by 20-fold relative to unmodified Cas9. This technology represents a new paradigm for biomolecular coupling, and paves the way to an unprecedented range of multifunctional bioconjugates.

Funding

CHE-1808189

GM68933

K99GM118909

Mechanism and Therapeutic Application of RNA-Guided Immune Systems

National Institute of General Medical Sciences

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Center for Genome Editing and Recording

National Human Genome Research Institute

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U01AI142817-02

Mechanism of Acquired Immunity in Bacteria

Directorate for Biological Sciences

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History

Email Address of Submitting Author

mbfrancis@berkeley.edu

Institution

UC Berkeley

Country

USA

ORCID For Submitting Author

0000-0003-2837-2538

Declaration of Conflict of Interest

J.A.D. is an Investigator of the Howard Hughes Medical Institute and executive director of the Innovative Genomics Institute at the University of California, Berkeley and the University of California, San Francisco. J.A.D. is a co-founder of Editas Medicine, Intellia Therapeutics, and Caribou Biosciences and a scientific adviser to Caribou, Intellia, eFFECTOR Therapeutics and Driver. The Regents of the University of California have patents pending for CRISPR technologies and the use of tyrosinase in the abovementioned manner on which the authors are inventors

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