Singh, Advaita APillay, PriyenNaicker, PrevinAlexandre, Kabamba BMalatji, KanyaneMach, LSteinkellner, HVorster, JChikwamba, Rachel KTsekoa, Tsepo L2022-10-032022-10-032022-08Singh, A.A., Pillay, P., Naicker, P., Alexandre, K.B., Malatji, K., Mach, L., Steinkellner, H. & Vorster, J. et al. 2022. Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9. <i>Frontiers in Plant Science, 13.</i> http://hdl.handle.net/10204/12501DOI: 10.3389/fpls.2022.953654http://hdl.handle.net/10204/12501The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant XTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and XTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors.FulltextenNicotiana benthamianaGenome editingHuman immunodeficiency virusImmunoglobulin GPlant biotechnologyProteasesArticleSingh, A. A., Pillay, P., Naicker, P., Alexandre, K. B., Malatji, K., Mach, L., ... Tsekoa, T. L. (2022). Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9. <i>Frontiers in Plant Science, 13</i>, http://hdl.handle.net/10204/12501Singh, Advaita A, Priyen Pillay, Previn Naicker, Kabamba B Alexandre, Kanyane Malatji, L Mach, H Steinkellner, J Vorster, Rachel K Chikwamba, and Tsepo L Tsekoa "Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9." <i>Frontiers in Plant Science, 13</i> (2022) http://hdl.handle.net/10204/12501Singh AA, Pillay P, Naicker P, Alexandre KB, Malatji K, Mach L, et al. Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9. Frontiers in Plant Science, 13. 2022; http://hdl.handle.net/10204/12501.TY - Article AU - Singh, Advaita A AU - Pillay, Priyen AU - Naicker, Previn AU - Alexandre, Kabamba B AU - Malatji, Kanyane AU - Mach, L AU - Steinkellner, H AU - Vorster, J AU - Chikwamba, Rachel K AU - Tsekoa, Tsepo L AB - The hypersensitive response is elicited by Agrobacterium infiltration of Nicotiana benthamiana, including the induction and accumulation of pathogenesis-related proteins, such as proteases. This includes the induction of the expression of several cysteine proteases from the C1 (papain-like cysteine protease) and C13 (legumain-like cysteine protease) families. This study demonstrates the role of cysteine proteases: NbVPE-1a, NbVPE-1b, and NbCysP6 in the proteolytic degradation of Nicotiana benthamiana (glycosylation mutant XTFT)-produced anti-human immunodeficiency virus broadly neutralizing antibody, CAP256-VRC26.25. Three putative cysteine protease cleavage sites were identified in the fragment crystallizable region. We further demonstrate the transient coexpression of CAP256-VRC26.25 with CRISPR/Cas9-mediated genome editing vectors targeting the NbVPE-1a, NbVPE-1b, and NbCysP6 genes which resulted in a decrease in CAP256-VRC26.25 degradation. No differences in structural features were observed between the human embryonic kidney 293 (HEK293)-produced and XTFT broadly neutralizing antibodies produced with and without the coexpression of genome-editing vectors. Furthermore, despite the presence of proteolytically degraded fragments of plant-produced CAP256-VRC26.25 without the coexpression of genome editing vectors, no influence on the in vitro functional activity was detected. Collectively, we demonstrate an innovative in planta strategy for improving the quality of the CAP256 antibodies through the transient expression of the CRISPR/Cas9 vectors. DA - 2022-08 DB - ResearchSpace DP - CSIR J1 - Frontiers in Plant Science, 13 KW - Nicotiana benthamiana KW - Genome editing KW - Human immunodeficiency virus KW - Immunoglobulin G KW - Plant biotechnology KW - Proteases LK - https://researchspace.csir.co.za PY - 2022 T1 - Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9 TI - Transient proteolysis reduction of Nicotiana benthamiana-produced CAP256 broadly neutralizing antibodies using CRISPR/Cas9 UR - http://hdl.handle.net/10204/12501 ER -25997CRISPR/Cas9