TY - JOUR AB - Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate. AD - Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States. Duncan Neurological Research Institute, Texas Children Hospital, Houston, United States. Department of Genetics, Harvard Medical School, Boston, United States. Department of Embryology, Carnegie Institution for Science, Baltimore, United States. Howard Hughes Medical Institute, Harvard Medical School, Boston, United States. Department of Neuroscience, Baylor College of Medicine, Houston, United States. AN - 35723254 AU - Kanca, O. AU - Zirin, J. AU - Hu, Y. AU - Tepe, B. AU - Dutta, D. AU - Lin, W. W. AU - Ma, L. AU - Ge, M. AU - Zuo, Z. AU - Liu, L. P. AU - Levis, R. W. AU - Perrimon, N. AU - Bellen, H. J. C1 - OK, JZ, YH, BT, DD, WL, LM, MG, ZZ, LL, RL, NP, HB No competing interests declared C2 - PMC9239680 DA - Jun 20 DO - 10.7554/eLife.76077 DP - NLM ET - 20220620 KW - Animals CRISPR-Cas Systems/genetics *Clustered Regularly Interspaced Short Palindromic Repeats/genetics *Drosophila/genetics Exons/genetics Homologous Recombination Plasmids Crispr D. melanogaster gene trap genetics genomics knock-in knock-out protein trap LA - eng N1 - 2050-084x Kanca, Oguz Orcid: 0000-0001-5438-0879 Zirin, Jonathan Hu, Yanhui Tepe, Burak Dutta, Debdeep Lin, Wen-Wen Ma, Liwen Ge, Ming Zuo, Zhongyuan Liu, Lu-Ping Levis, Robert W Orcid: 0000-0003-3453-2390 Perrimon, Norbert Orcid: 0000-0001-7542-472x Bellen, Hugo J Orcid: 0000-0001-5992-5989 U54 HD083092/HD/NICHD NIH HHS/United States R01 GM067761/GM/NIGMS NIH HHS/United States R01 GM067858/GM/NIGMS NIH HHS/United States R01 GM084947/GM/NIGMS NIH HHS/United States HHMI/Howard Hughes Medical Institute/United States R24 OD031447/OD/NIH HHS/United States U54 NS093793/NS/NINDS NIH HHS/United States R24 OD022005/OD/NIH HHS/United States Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't England 2022/06/21 Elife. 2022 Jun 20;11:e76077. doi: 10.7554/eLife.76077. PY - 2022 SN - 2050-084x ST - An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination T2 - Elife TI - An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination VL - 11 ID - 4649 ER - TY - JOUR AB - PURPOSE: Growth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants. METHODS: We present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality. RESULTS: Patients with variants in GDF11 presented with craniofacial (5/6), vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6), and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients' variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) allele whereas the missense variants in our cohort are partial LOF variants. CONCLUSION: GDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues. AD - Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA. Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX, USA. Institute of Neuroscience, University of Oregon, Eugene, OR, USA. Brigham and Women's Hospital, Boston, MA, USA. Baylor Genetics Laboratories, Houston, TX, USA. Department of Genetics, Harvard Medical School, Boston, MA, USA. Joe DiMaggio Children's Hospital, Hollywood, FL, USA. Institute of Human Genetics, University of Leipzig Medical Center, Leipzig, Germany. Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada. The Chinese University of Hong Kong-Baylor College of Medicine Joint Center of Medical Genetics, Hong Kong Special Administrative Region, The People's Republic of China. Boston Children's Hospital, Boston, MA, USA. Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA. hbellen@bcm.edu. Jan and Dan Duncan Neurological Research Institute, Texas Children Hospital, Houston, TX, USA. hbellen@bcm.edu. Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA. hbellen@bcm.edu. Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX, USA. hbellen@bcm.edu. AN - 34113007 AU - Ravenscroft, T. A. AU - Phillips, J. B. AU - Fieg, E. AU - Bajikar, S. S. AU - Peirce, J. AU - Wegner, J. AU - Luna, A. A. AU - Fox, E. J. AU - Yan, Y. L. AU - Rosenfeld, J. A. AU - Zirin, J. AU - Kanca, O. AU - Benke, P. J. AU - Cameron, E. S. AU - Strehlow, V. AU - Platzer, K. AU - Jamra, R. A. AU - Klöckner, C. AU - Osmond, M. AU - Licata, T. AU - Rojas, S. AU - Dyment, D. AU - Chong, J. S. C. AU - Lincoln, S. AU - Stoler, J. M. AU - Postlethwait, J. H. AU - Wangler, M. F. AU - Yamamoto, S. AU - Krier, J. AU - Westerfield, M. AU - Bellen, H. J. C1 - Declaration of interests The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing conducted at Baylor Genetics Laboratories. The authors have no other conflicts of interest. C2 - PMC8487929 C6 - NIHMS1717830 DA - Oct DO - 10.1038/s41436-021-01216-8 DP - NLM ET - 20210610 IS - 10 KW - Animals *Bone Morphogenetic Proteins/genetics Craniofacial Abnormalities/*genetics *Growth Differentiation Factors/genetics Humans Mutation, Missense Phenotype Spine Zebrafish/genetics LA - eng N1 - 1530-0366 Ravenscroft, Thomas A Phillips, Jennifer B Fieg, Elizabeth Bajikar, Sameer S Peirce, Judy Wegner, Jeremy Luna, Alia A Fox, Eric J Yan, Yi-Lin Rosenfeld, Jill A Zirin, Jonathan Kanca, Oguz Undiagnosed Diseases Network Benke, Paul J Cameron, Eric S Strehlow, Vincent Platzer, Konrad Jamra, Rami Abou Klöckner, Chiara Osmond, Matthew Licata, Thomas Rojas, Samantha Dyment, David Chong, Josephine S C Lincoln, Sharyn Stoler, Joan M Postlethwait, John H Wangler, Michael F Yamamoto, Shinya Krier, Joel Westerfield, Monte Bellen, Hugo J U01 HG007690/HG/NHGRI NIH HHS/United States P41 GM132087/GM/NIGMS NIH HHS/United States R24 OD031447/OD/NIH HHS/United States F32 HD100048/HD/NICHD NIH HHS/United States U01 HG007942/HG/NHGRI NIH HHS/United States R24 OD026591/OD/NIH HHS/United States U54 NS093793/NS/NINDS NIH HHS/United States R24 OD022005/OD/NIH HHS/United States Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't United States 2021/06/12 Genet Med. 2021 Oct;23(10):1889-1900. doi: 10.1038/s41436-021-01216-8. Epub 2021 Jun 10. PY - 2021 SN - 1098-3600 (Print) 1098-3600 SP - 1889-1900 ST - Heterozygous loss-of-function variants significantly expand the phenotypes associated with loss of GDF11 T2 - Genet Med TI - Heterozygous loss-of-function variants significantly expand the phenotypes associated with loss of GDF11 VL - 23 ID - 4650 ER -