Endogenous tagging of multiple cellular components in the sea anemone Nematostella vectensis.

The cnidarian Nematostella vectensis has developed into a powerful model system to study the mechanisms underlying animal development, regeneration, and evolution. However, despite the significant progress in the molecular and genetic approaches in this sea anemone, endogenous protein tagging is still challenging. Here, we report a robust method for knock in for Nematostella using CRISPR/Cas9. As an outcome, we generate endogenously tagged proteins that label core molecular components of several cellular apparatus, including the nuclear envelope, cytoskeleton, cell adhesion, endoplasmic reticulum, cell trafficking, and extracellular matrix. Using live imaging, we monitor the dynamics of vesicular trafficking and endoplasmic reticulum in embryos, as well as cell contractility during the peristaltic wave of a primary polyp. This advancement in gene editing expands the molecular tool kit of Nematostella and enables experimental avenues to interrogate the cell biology of cnidarians.

Oblique plane microscope for mesoscopic imaging of freely moving organisms with cellular resolution.

Several important questions in biology require non-invasive and three-dimensional imaging techniques with an appropriate spatiotemporal resolution that permits live organisms to move in an unconstrained fashion over an extended field-of-view. While selective-plane illumination microscopy (SPIM) has emerged as a powerful method to observe live biological specimens at high spatio-temporal resolution, typical implementations often necessitate constraining sample mounting or lack the required volumetric speed. Here, we report on an open-top, dual-objective oblique plane microscope (OPM) capable of observing millimeter-sized, freely moving animals at cellular resolution. We demonstrate the capabilities of our mesoscopic OPM (MesOPM) by imaging the behavioral dynamics of the sea anemone Nematostella vectensis over 1.56 × 1.56 × 0.25 mm at 1.5 × 2.8 × 5.3 µm resolution and 0.5 Hz volume rate.

Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks.

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double stranded) engage in a high-efficiency HDR mechanism that requires only ∼35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1,000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand annealing. Our findings enable rational design of synthetic donor DNAs for efficient genome editing.

Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans.

The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleoprotein complexes and use of linear DNAs with short homology arms as repair templates. The protocol requires no cloning or selection, and can be used to generate base and gene-size edits in just 4days. Point mutations, insertions, deletions and gene replacements can all be created using the same experimental pipeline.

Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs.

Recombineering, the use of endogenous homologous recombination systems to recombine DNA in vivo, is a commonly used technique for genome editing in microbes. Recombineering has not yet been developed for animals, where non-homology-based mechanisms have been thought to dominate DNA repair. Here, we demonstrate, using Caenorhabditis elegans, that linear DNAs with short homologies (∼35 bases) engage in a highly efficient gene conversion mechanism. Linear DNA repair templates with homology to only one side of a double-strand break (DSB) initiate repair efficiently, and short overlaps between templates support template switching. We demonstrate the use of single-stranded, bridging oligonucleotides (ssODNs) to target PCR fragments for repair of DSBs induced by CRISPR/Cas9 on chromosomes. Based on these findings, we develop recombineering strategies for precise genome editing that expand the utility of ssODNs and eliminate in vitro cloning steps for template construction. We apply these methods to the generation of GFP knock-in alleles and gene replacements without co-integrated markers. We conclude that, like microbes, metazoans possess robust homology-dependent repair mechanisms that can be harnessed for recombineering and genome editing.

The P granule component PGL-1 promotes the localization and silencing activity of the PUF protein FBF-2 in germline stem cells.

In the C. elegans germline, maintenance of undifferentiated stem cells depends on the PUF family RNA-binding proteins FBF-1 and FBF-2. FBF-1 and FBF-2 are 89% identical and are required redundantly to silence the expression of mRNAs that promote meiosis. Here we show that, despite their extensive sequence similarity, FBF-1 and FBF-2 have different effects on target mRNAs. FBF-1 promotes the degradation and/or transport of meiotic mRNAs out of the stem cell region, whereas FBF-2 prevents translation. FBF-2 activity depends on the P granule component PGL-1. PGL-1 is required to localize FBF-2 to perinuclear P granules and for efficient binding of FBF-2 to its mRNA targets. We conclude that multiple regulatory mechanisms converge on meiotic RNAs to ensure silencing in germline stem cells. Our findings also support the view that P granules facilitate mRNA silencing by providing an environment in which translational repressors can encounter their mRNA targets immediately upon exit from the nucleus.

Drosophila ALS regulates growth and metabolism through functional interaction with insulin-like peptides.

In metazoans, factors of the insulin family control growth, metabolism, longevity, and fertility in response to environmental cues. In Drosophila, a family of seven insulin-like peptides, called Dilps, activate a common insulin receptor. Some Dilp peptides carry both metabolic and growth functions, raising the possibility that various binding partners specify their functions. Here we identify dALS, the fly ortholog of the vertebrate insulin-like growth factor (IGF)-binding protein acid-labile subunit (ALS), as a Dilp partner that forms a circulating trimeric complex with one molecule of Dilp and one molecule of Imp-L2, an IgG-family molecule distantly related to mammalian IGF-binding proteins (IGFBPs). We further show that dALS antagonizes Dilp function to control animal growth as well as carbohydrate and fat metabolism. These results lead us to propose an evolutionary perspective in which ALS function appeared prior to the separation between metabolic and growth effects that are associated with vertebrate insulin and IGFs.

Bi-polarized translation of ascidian maternal mRNA determinant pem-1 associated with regulators of the translation machinery on cortical Endoplasmic Reticulum (cER).

Polarized cortical mRNA determinants such as maternal macho-1 and pem-1 in ascidians, like budding yeast mating factor ASH1 reside on the cER-mRNA domain a subdomain of cortical Endoplasmic Reticulum(ER) and are translated in its vicinity. Using high resolution imaging and isolated cortical fragments prepared from eggs and embryos we now find that macho-1 and pem-1 RNAs co-localize with phospho-protein regulators of translation initiation (MnK/4EBP/S6K). Translation of cortical pem-1 RNA follows its bi-polarized relocalization. About 10 min after fertilization or artificial activation with a calcium ionophore, PEM1 protein is detected in the vegetal cortex in the vicinity of pem-1 RNA. About 40 min after fertilization-when pem-1 RNA and P-MnK move to the posterior pole-PEM1 protein remains in place forming a network of cortical patches anchored at the level of the zygote plasma membrane before disappearing. Cortical PEM1 protein is detected again at the 4 cell stage in the posterior centrosome attracting body (CAB) region where the cER-mRNA domain harboring pem-1/P-MnK/P-4EBP/P-S6K is concentrated. Bi-polarized PEM1 protein signals are not detected when pem-1 morpholinos are injected into eggs or zygotes or when MnK is inhibited. We propose that localized translation of the pem-1 RNA determinant is triggered by the fertilization/calcium wave and that the process is controlled by phospho-protein regulators of translation initiation co-localized with the RNA determinant on a sub-domain of the cortical Endoplasmic Reticulum.

Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach.

The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology-directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient, and precise strategy for CRISPR/Cas9-mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR-amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40 bp), a synthetic single-guide RNA and Cas9 mRNA. We generate eight novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole genome sequencing results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.

Cortical anchorages and cell type segregations of maternal postplasmic/PEM RNAs in ascidians.

Ascidian postplasmic/PEM RNAs constitute a large class of cortical maternal RNAs which include developmental determinants (macho-1 and pem-1). We have analyzed the localization, cortical anchorage and cell type segregation of postplasmic/PEM RNAs in Ciona intestinalis and Phallusia mammillata using very high-resolution fluorescent in situ hybridization. We also compared RNAs extracted from whole oocytes and from isolated cortices using microarrays and localized RNAs possessing clusters of xCACx motifs in their 3'UTRs. Based on these combined approaches we conclude that: (1) the vast majority of the 39 postplasmic/PEM RNAs (including vasa) are localized in the egg cortex. (2) Many postplasmic/PEM RNAs 3'UTR are enriched in xCACx motifs, allowing us to identify 2 novel postplasmic/PEM RNAs (PSD and MnK). (3) Postplasmic/PEM RNAs anchored to cortical Endoplasmic Reticulum (cER) and those associated with granules have different cell destinations. We propose that there are 2 distinct categories of postplasmic/PEM RNAs on the basis of their cortical anchorages and cell destinations: (1) macho-1-like postplasmic/PEM RNAs anchored to cER which segregate into somatic B8.11 cells. (2) vasa-like postplasmic/PEM RNAs associated with granules which in addition to B8.11 cells segregate into B8.12 germ cells.

Rapid Tagging of Human Proteins with Fluorescent Reporters by Genome Engineering using Double-Stranded DNA Donors.

Tagging proteins with fluorescent reporters such as green fluorescent protein (GFP) is a powerful method to determine protein localization, especially when proteins are tagged in the endogenous context to preserve native genomic regulation. However, insertion of fluorescent reporters into the genomes of mammalian cells has required the construction of plasmids containing selection markers and/or extended sequences homologous to the site of insertion (homology arms). Here we describe a streamlined protocol that eliminates all cloning steps by taking advantage of the high propensity of linear DNAs to engage in homology-directed repair of DNA breaks induced by the Cas9 RNA-guided endonuclease. The protocol uses PCR amplicons, or synthetic gene fragments, with short homology arms (30-40 bp) to insert fluorescent reporters at specific genomic locations. The linear DNAs are introduced into cells with preassembled Cas9-crRNA-tracrRNA complexes using one of two transfection procedures, nucleofection or lipofection. The protocol can be completed under a week, with efficiencies ranging from 0.5% to 20% of transfected cells depending on the locus targeted. © 2019 The Authors.

High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes.

Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.

Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans.

Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (∼30-60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.

Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans.

RNA granules have been likened to liquid droplets whose dynamics depend on the controlled dissolution and condensation of internal components. The molecules and reactions that drive these dynamics in vivo are not well understood. In this study, we present evidence that a group of intrinsically disordered, serine-rich proteins regulate the dynamics of P granules in C. elegans embryos. The MEG (maternal-effect germline defective) proteins are germ plasm components that are required redundantly for fertility. We demonstrate that MEG-1 and MEG-3 are substrates of the kinase MBK-2/DYRK and the phosphatase PP2A(PPTR-½). Phosphorylation of the MEGs promotes granule disassembly and dephosphorylation promotes granule assembly. Using lattice light sheet microscopy on live embryos, we show that GFP-tagged MEG-3 localizes to a dynamic domain that surrounds and penetrates each granule. We conclude that, despite their liquid-like behavior, P granules are non-homogeneous structures whose assembly in embryos is regulated by phosphorylation.

Localization and anchorage of maternal mRNAs to cortical structures of ascidian eggs and embryos using high resolution in situ hybridization.

In several species, axis formation and tissue differentiation are the result of developmental cascades which begin with the localization and translation of key maternal mRNAs in eggs. Localization and anchoring of mRNAs to cortical structures can be observed with high sensitivity and resolution by fluorescent in situ hybridization coupled with labeling of membranes and macromolecular complexes. Oocytes and embryos of ascidians--marine chordates closely related to vertebrates--are ideal models to understand how maternal mRNAs pattern the simple ascidian tadpole. More than three dozen cortically localized maternal mRNAs have been identified in ascidian eggs. They include germ cell markers such as vasa or pem-3 and determinants of axis (pem-1), unequal cleavage (pem-1), and muscle cells (macho-1). High resolution localization of mRNAs, proteins, and lipids in whole eggs and embryos and their cortical fragments shows that maternal mRNA determinants pem-1 and macho-1 are anchored to cortical endoplasmic reticulum and segregate with it into small posterior somatic cells. In contrast, mRNAs such as vasa are associated with granular structures which are inherited by the same somatic cells plus adjacent germ cell precursors. In this chapter, we provide detailed protocols for simultaneous localization of mRNAs and proteins to determine their association with cellular structures in eggs and embryos. Using preparations of isolated cortical fragments with intact membranous structures allows unprecedented high resolution analysis and identification of cellular anchoring sites for key mRNAs. This information is necessary for understanding the mechanisms for localizing mRNAs and partitioning them into daughter cells after cleavage.

Embryological methods in ascidians: the Villefranche-sur-Mer protocols.

Ascidians (marine invertebrates: urochordates) are thought to be the closest sister groups of vertebrates. They are particularly attractive models because of their non-duplicated genome and the fast and synchronous development of large populations of eggs into simple tadpoles made of about 3,000 cells. As a result of stereotyped asymmetric cleavage patterns all blastomeres become fate restricted between the 16- and 110 cell stage through inheritance of maternal determinants and/or cellular interactions. These advantageous features have allowed advances in our understanding of the nature and role of maternal determinants, inductive interactions, and gene networks that are involved in cell lineage specification and differentiation of embryonic tissues. Ascidians have also contributed to our understanding of fertilization, cell cycle control, self-recognition, metamorphosis, and regeneration. In this chapter we provide basic protocols routinely used at the marine station in Villefranche-sur-Mer using the cosmopolitan species of reference Ciona intestinalis and the European species Phallusia mammillata. These two models present complementary advantages with regard to molecular, functional, and imaging approaches. We describe techniques for basic culture of embryos, micro-injection, in vivo labelling, micro-manipulations, fixation, and immuno-labelling. These methods allow analysis of calcium signals, reorganizations of cytoplasmic and cortical domains, meiotic and mitotic cell cycle and cleavages as well as the roles of specific genes and cellular interactions. Ascidians eggs and embryos are also an ideal material to isolate cortical fragments and to isolate and re-associate individual blastomeres. We detail the experimental manipulations which we have used to understand the structure and role of the egg cortex and of specific blastomeres during development.

From oocyte to 16-cell stage: cytoplasmic and cortical reorganizations that pattern the ascidian embryo.

The dorsoventral and anteroposterior axes of the ascidian embryo are defined before first cleavage by means of a series of reorganizations that reposition cytoplasmic and cortical domains established during oogenesis. These domains situated in the periphery of the oocyte contain developmental determinants and a population of maternal postplasmic/PEM RNAs. One of these RNAs (macho-1) is a determinant for the muscle cells of the tadpole embryo. Oocytes acquire a primary animal-vegetal (a-v) axis during meiotic maturation, when a subcortical mitochondria-rich domain (myoplasm) and a domain rich in cortical endoplasmic reticulum (cER) and maternal postplasmic/PEM RNAs (cER-mRNA domain) become polarized and asymmetrically enriched in the vegetal hemisphere. Fertilization at metaphase of meiosis I initiates a series of dramatic cytoplasmic and cortical reorganizations of the zygote, which occur in two major phases. The first major phase depends on sperm entry which triggers a calcium wave leading in turn to an actomyosin-driven contraction wave. The contraction concentrates the cER-mRNA domain and myoplasm in and around a vegetal/contraction pole. The precise localization of the vegetal/contraction pole depends on both the a-v axis and the location of sperm entry and prefigures the future site of gastrulation and dorsal side of the embryo. The second major phase of reorganization occurs between meiosis completion and first cleavage. Sperm aster microtubules and then cortical microfilaments cause the cER-mRNA domain and myoplasm to reposition toward the posterior of the zygote. The location of the posterior pole depends on the localization of the sperm centrosome/aster attained during the first major phase of reorganization. Both cER-mRNA and myoplasm domains localized in the posterior region are partitioned equally between the first two blastomeres and then asymmetrically over the next two cleavages. At the eight-cell stage the cER-mRNA domain compacts and gives rise to a macroscopic cortical structure called the Centrosome Attracting Body (CAB). The CAB is responsible for a series of unequal divisions in posterior-vegetal blastomeres, and the postplasmic/PEM RNAs it contains are involved in patterning the posterior region of the embryo. In this review, we discuss these multiple events and phases of reorganizations in detail and their relationship to physiological, cell cycle, and cytoskeletal events. We also examine the role of the reorganizations in localizing determinants, postplasmic/PEM RNAs, and PAR polarity proteins in the cortex. Finally, we summarize some of the remaining questions concerning polarization of the ascidian embryo and provide comparisons to a few other species. A large collection of films illustrating the reorganizations can be consulted by clicking on "Film archive: ascidian eggs and embryos" at http://biodev.obs-vlfr.fr/recherche/biomarcell/.

The aPKC-PAR-6-PAR-3 cell polarity complex localizes to the centrosome attracting body, a macroscopic cortical structure responsible for asymmetric divisions in the early ascidian embryo.

Posterior blastomeres of 8-cell stage ascidian embryos undergo a series of asymmetric divisions that generate cells of unequal sizes and segregate muscle from germ cell fates. These divisions are orchestrated by a macroscopic cortical structure, the ;centrosome attracting body' (CAB) which controls spindle positioning and distribution of mRNA determinants. The CAB is composed of a mass of cortical endoplasmic reticulum containing mRNAs (the cER-mRNA domain) and an electron dense matrix, but little is known about its precise structure and functions. We have examined the ascidian homologues of PAR proteins, known to regulate polarity in many cell types. We found that aPKC, PAR-6 and PAR-3 proteins, but not their mRNAs, localize to the CAB during the series of asymmetric divisions. Surface particles rich in aPKC concentrate in the CAB at the level of cortical actin microfilaments and form a localized patch sandwiched between the plasma membrane and the cER-mRNA domain. Localization of aPKC to the CAB is dependent on actin but not microtubules. Both the aPKC layer and cER-mRNA domain adhere to cortical fragments prepared from 8-cell stage embryos. Astral microtubules emanating from the proximal centrosome contact the aPKC-rich cortical domain. Our observations indicate that asymmetric division involves the accumulation of the aPKC-PAR-6-PAR-3 complex at the cortical position beneath the pre-existing cER-mRNA domain.