G3 (Bethesda, Md.)
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Chromatin-remodeling proteins have a profound role in the transcriptional regulation of gene expression during development. Here, we have shown that the chromodomain-containing protein Hat-trick is predominantly expressed within the oocyte nucleus, specifically within the heterochromatinized karyosome, and that a mild expression is observed in follicle cells. Colocalization of Hat-trick with Heterochromatin Protein 1 and synaptonemal complex component C(3)G along with the diffused karyosome after hat-trick downregulation shows the role of this protein in heterochromatin clustering and karyosome maintenance. ⋯ The increased incidence of double-strand breaks (DSBs), delayed DSB repair, defects in karyosome formation, altered Vasa mobility, and, consequently, misexpression and altered localization of Gurken in hat-trick mutant egg chambers clearly suggest a putative involvement of Hat-trick in the early stages of oogenesis. In addition, based on phenotypic observations in hat-trick mutant egg chambers, we speculate a substantial role of hat-trick in cystoblast proliferation, oocyte determination, nurse cell endoreplication, germ cell positioning, cyst encapsulation, and nurse cell migration. Our results demonstrate that hat-trick has profound pleiotropic functions during oogenesis in Drosophila melanogaster.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. ⋯ Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system.