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Jobst-Schwan, Tilman

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Jobst-Schwan

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Tilman

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Jobst-Schwan, Tilman
Author's Publications: search.filters.isAuthorOfPublication.5055752a-2d31-40b8-904d-0cef99b4a379

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    Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model
    (Public Library of Science, 2018) Jobst-Schwan, Tilman; Schmidt, Johanna Magdalena; Schneider, Ronen; Hoogstraten, Charlotte A.; Ullmann, Jeremy; Schapiro, David; Majmundar, Amar; Kolb, Amy; Eddy, Kaitlyn; Shril, Shirlee; Braun, Daniela A.; Poduri, Annapurna; Hildebrandt, Friedhelm
    Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest.
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    Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment
    (Nature Publishing Group UK, 2018) Ashraf, Shazia; Kudo, Hiroki; Rao, Jia; Kikuchi, Atsuo; Widmeier, Eugen; Lawson, Jennifer A.; Tan, Weizhen; Hermle, Tobias; Warejko, Jillian K.; Shril, Shirlee; Airik, Merlin; Jobst-Schwan, Tilman; Lovric, Svjetlana; Braun, Daniela A.; Gee, Heon Yung; Schapiro, David; Majmundar, Amar; Sadowski, Carolin E.; Pabst, Werner L.; Daga, Ankana; van der Ven, Amelie T.; Schmidt, Johanna M.; Low, Boon Chuan; Gupta, Anjali Bansal; Tripathi, Brajendra K.; Wong, Jenny; Campbell, Kirk; Metcalfe, Kay; Schanze, Denny; Niihori, Tetsuya; Kaito, Hiroshi; Nozu, Kandai; Tsukaguchi, Hiroyasu; Tanaka, Ryojiro; Hamahira, Kiyoshi; Kobayashi, Yasuko; Takizawa, Takumi; Funayama, Ryo; Nakayama, Keiko; Aoki, Yoko; Kumagai, Naonori; Iijima, Kazumoto; Fehrenbach, Henry; Kari, Jameela A.; El Desoky, Sherif; Jalalah, Sawsan; Bogdanovic, Radovan; Stajić, Nataša; Zappel, Hildegard; Rakhmetova, Assel; Wassmer, Sharon-Rose; Jungraithmayr, Therese; Strehlau, Juergen; Kumar, Aravind Selvin; Bagga, Arvind; Soliman, Neveen A.; Mane, Shrikant M.; Kaufman, Lewis; Lowy, Douglas R.; Jairajpuri, Mohamad A.; Lifton, Richard P.; Pei, York; Zenker, Martin; Kure, Shigeo; Hildebrandt, Friedhelm
    No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes (MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2, DLC1, or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2-L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets.