ORCID Profile
0000-0003-3924-3635
Current Organisation
Scuola Normale Superiore
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Publisher: Wiley
Date: 26-06-2013
DOI: 10.1111/JCMM.12085
Publisher: Cold Spring Harbor Laboratory
Date: 18-04-2017
DOI: 10.1101/127506
Abstract: The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economical impact and has become an emerging model for developing new genetic control strategies as alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific sgRNAs into early embryos. When targeting the eye pigmentation gene white eye ( we ), we observed a high rate of somatic mosaicism in surviving G0 adults. Germline transmission of mutated we alleles by G0 animals was on average above 70%, with in idual cases achieving a transmission rate of nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene ( Ccprd ) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in Ceratitis capitata will significantly advance the design and development of new effective strategies for pest control management.
Publisher: Springer Science and Business Media LLC
Date: 30-08-2017
DOI: 10.1038/S41598-017-10347-5
Abstract: The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye ( we ), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with in idual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene ( Ccprd ) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.
Publisher: Springer Science and Business Media LLC
Date: 07-2016
DOI: 10.1038/NBT.3628
Publisher: The Company of Biologists
Date: 2016
DOI: 10.1242/DEV.134809
Abstract: CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro-assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in in idual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene-regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.
Publisher: Cold Spring Harbor Laboratory
Date: 12-04-2023
DOI: 10.1101/2023.04.12.536513
Abstract: Defects in blood development frequently occur among syndromic congenital anomalies. Thrombocytopenia-Absent Radius (TAR) Syndrome is a rare congenital condition with reduced platelets (hypomegakaryocytic thrombocytopenia) and forelimb anomalies, concurrent with more variable heart and kidney defects. TAR syndrome associates with hypomorphic gene function for RBM8A/Y14 that encodes a component of the exon junction complex involved in mRNA splicing, transport, and nonsense-mediated decay. How perturbing a general mRNA-processing factor causes the selective TAR Syndrome phenotypes remains unknown. Here, we connect zebrafish rbm8a perturbation to early hematopoietic defects via attenuated non-canonical Wnt/Planar Cell Polarity (PCP) signaling that controls developmental cell arrangements. In hypomorphic rbm8a zebrafish, we observe a significant reduction of cd41 -positive thrombocytes. rbm8a -mutant zebrafish embryos accumulate mRNAs with in idual retained introns, a hallmark of defective nonsense-mediated decay affected mRNAs include transcripts for non-canonical Wnt/PCP pathway components. We establish that rbm8a -mutant embryos show convergent extension defects and that reduced rbm8a function interacts with perturbations in non-canonical Wnt/PCP pathway genes w nt5b , wnt11f2 , fzd7a , and vangl2 . Using live-imaging, we found reduced rbm8a function impairs the architecture of the lateral plate mesoderm (LPM) that forms hematopoietic, cardiovascular, kidney, and forelimb skeleton progenitors as affected in TAR Syndrome. Both mutants for rbm8a and for the PCP gene vangl2 feature impaired expression of early hematopoietic/endothelial genes including runx1 and the megakaryocyte regulator gfi1aa . Together, our data propose aberrant LPM patterning and hematopoietic defects as possible consequence of attenuated non-canonical Wnt/PCP signaling upon reduced rbm8a function. These results link TAR Syndrome to a potential LPM origin and developmental mechanism.
Location: United Kingdom of Great Britain and Northern Ireland
Location: Japan
No related grants have been discovered for Elena Chiavacci.