Elena Chiavacci

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

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-sgRNA 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 individual 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. Summary: Maximal mutagenesis efficiency is achieved in vivo in zebrafish embryos using salt-solubilized, fluorescently labelled Cas9-sgRNA complexes.

MicroRNA 19a replacement partially rescues fin and cardiac defects in zebrafish model of Holt Oram syndrome

Holt-Oram Syndrome (HOS) is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene, a transcription factor capable of regulating hundreds of cardiac-specific genes through complex transcriptional networks. Here we show that, in zebrafish, modulation of a single miRNA is sufficient to rescue the morphogenetic defects generated by HOS. The analysis of miRNA-seq profiling revealed a decreased expression of miR-19a in Tbx5-depleted zebrafish embryos compared to the wild type. We revealed that the transcription of the miR-17-92 cluster, which harbors miR-19a, is induced by Tbx5 and that a defined dosage of miR-19a is essential for the correct development of the heart. Importantly, we highlighted that miR-19a replacement is able to rescue cardiac and pectoral fin defects and to increase the viability of HOS zebrafish embryos. We further observed that miR-19a replacement shifts the global gene expression profile of HOS-like zebrafish embryos towards the wild type condition, confirming the ability of miR-19a to rescue the Tbx5 phenotype. In conclusion our data demonstrate the importance of Tbx5/miR-19a regulatory circuit in heart development and provide a proof of principle that morphogenetic defects associated with HOS can be rescued by transient miRNA modulation.

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes

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 individual 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.

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 RNPs

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 individual 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.

CrispRVariants: precisely charting the mutation spectrum in genome engineering experiments

CRISPR-Cas9 and related technologies efficiently alter genomic DNA at targeted positions and have far-reaching implications for functional screening and therapeutic gene editing. Understanding and unlocking this potential requires accurate evaluation of editing efficiency. We show that methodological decisions for analyzing sequencing data can significantly affect mutagenesis efficiency estimates and we provide a comprehensive R-based toolkit, CrispRVariants and accompanying web tool CrispRVariantsLite, that resolves and localizes individual mutant alleles with respect to the endonuclease cut site. CrispRVariants-enabled analyses of newly generated and existing genome editing datasets underscore how careful consideration of the full variant spectrum gives insight toward effective guide and amplicon design as well as the mutagenic process.

Cre/lox-controlled spatio-temporal perturbation of FGF signaling in zebrafish

Fibroblast Growth Factor (FGF) signaling guides multiple developmental processes including body axis formation and specific cell fate patterning. In zebrafish, genetic mutants and chemical perturbations affecting FGF signaling have uncovered key developmental processes; however, these approaches cause embryo-wide FGF signaling perturbations, rendering assessment of cell-autonomous versus non-autonomous requirements for FGF signaling in individual processes difficult. Here, we created the novel transgenic line fgfr1-dn-cargo, encoding dominant-negative Fgfr1 with fluorescent tag under combined Cre/lox and heatshock control to provide spatio-temporal perturbation of FGF signaling. Validating efficient perturbation of FGF signaling by fgfr1-dn-cargo primed with ubiquitous CreERT2, we established that primed, heatshock-induced fgfr1-dn-cargo behaves akin to pulsed treatment with the FGFR inhibitor SU5402. Priming fgfr1-dn-cargo with CreERT2 in the lateral plate mesoderm, we observed selective cardiac and pectoral fin phenotypes without drastic impact on overall embryo patterning. Harnessing lateral plate mesoderm-specific FGF inhibition, we recapitulated the cell-autonomous and temporal requirement for FGF signaling in pectoral fin outgrow, as previously hypothesized from pan-embryonic FGF inhibition. Altogether, our results establish fgfr1-dn-cargo as a genetic tool to define the spatio-temporal requirements for FGF signaling in zebrafish.

A conserved regulatory program drives emergence of the lateral plate mesoderm

Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolated a pan-LPM enhancer in the zebrafish draculin (drl) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl-based reporters captured the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncovered specific drl reporter activity in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona, and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.

Mutations in Bcl9 and Pygo genes cause congenital heart defects by tissue-specific perturbation of Wnt/β-catenin signaling

Bcl9 and Pygopus (Pygo) are obligate Wnt/β-catenin cofactors in Drosophila, yet their contribution to Wnt signaling during vertebrate development remains unresolved. Combining zebrafish and mouse genetics, we document a conserved, β-catenin-associated function for BCL9 and Pygo proteins during vertebrate heart development. Disrupting the β-catenin-BCL9-Pygo complex results in a broadly maintained canonical Wnt response yet perturbs heart development and proper expression of key cardiac regulators. Our work highlights BCL9 and Pygo as selective β-catenin cofactors in a subset of canonical Wnt responses during vertebrate development. Moreover, our results implicate alterations in BCL9 and BCL9L in human congenital heart defects.

Cre/lox-controlled spatiotemporal perturbation of FGF signaling in zebrafish: Controlling FGF Signaling in Zebrafish

Background: Spatiotemporal perturbation of signaling pathways in vivo remains challenging and requires precise transgenic control of signaling effectors. Fibroblast growth factor (FGF) signaling guides multiple developmental processes, including body axis formation and cell fate patterning. In zebrafish, mutants and chemical perturbations affecting FGF signaling have uncovered key developmental processes; however, these approaches cause embryo‐wide perturbations, rendering assessment of cell‐autonomous vs. non‐autonomous requirements for FGF signaling in individual processes difficult.