O. Kanca, J. Zirin, Y Hu, B. Tepe, D. Dutta, W. W Lin, L. Ma, M Ge, Z. Zuo, L. P Liu, R. W Levis, N. Perrimon, and H. J Bellen. Published. “
An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination.” Elife 11.
AbstractPreviously, 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.
T. A Ravenscroft, J. B Phillips, E. Fieg, S. S Bajikar, J. Peirce, J. Wegner, A. A Luna, E. J Fox, Y. L Yan, J. A Rosenfeld, J. Zirin, O. Kanca, P. J Benke, E. S Cameron, V. Strehlow, K. Platzer, R. A Jamra, C. Klöckner, M. Osmond, T. Licata, S. Rojas, D. Dyment, J. SC Chong, S. Lincoln, J. M Stoler, J. H Postlethwait, M. F Wangler, S. Yamamoto, J. Krier, M. Westerfield, and H. J Bellen. Published. “
Heterozygous loss-of-function variants significantly expand the phenotypes associated with loss of GDF11.” Genet Med, 23, 10, Pp. 1889-1900.
AbstractPURPOSE: 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.