Gene Knockdown Technology Research Articles

Ribosomal S6 kinase 2-forkhead box protein O4 signaling pathway plays an essential role in melanogenesis

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4’s transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.

Knockout of CLTC gene reduces but not completely block SFTSV infection.

Clathrin is a key protein for viruses to enter host cells. Previous studies often use clathrin inhibitors or gene knockdown technology to partially inhibit the function of clathrin, but whether SFTSV can infect host cells without clathrin expression remains unclear. In this research, a clathrin heavy chains (CLTC) knockout A549 cell line was established by CRISPR/Cas9 technology, and the knockout of CLTC was verified by PCR, Western blot, immunofluorescence and T7E1 analysis. The off-target effect was evaluated by PCR combined with Sanger sequencing. Furthermore, this research verified that SFTSV infection was significantly inhibited, but not completely blocked, due to the deletion of CLTC protein. Our research also found that lipid raft inhibitor Filipin, other than macropinocytosis inhibitor EIPA, could significantly reduce SFTSV infection, and the inhibition was more obviously observed when Filipin was used in CLTC knockout cells. These result indicated that clathrin-dependent and lipid raft mediated endocytosis are the major two mode used by SFTSV entry. In conclusion, this study constructed a CLTC knockout cell line, which, for the first time, established a cell model for the study of the function of CLTC protein, and provided direct evidence that SFTSV pendent could still infect cells without clathrin. Additionally, we confirmed that lipid raft mediated endocytosis, as a clathrin-independent pathway, could be another key mode for SFTSV entry.

YULINK regulates vascular formation in zebrafish and HUVECs

BackgroundThe distinct arterial and venous cell fates are dictated by a combination of various genetic factors which form diverse types of blood vessels such as arteries, veins, and capillaries. We report here that YULINK protein is involved in vasculogenesis, especially venous formation.MethodsIn this manuscript, we employed gene knockdown, yeast two-hybrid, FLIM-FRET, immunoprecipitation, and various imaging technologies to investigate the role of YULINK gene in zebrafish and human umbilical vein endothelial cells (HUVECs).ResultsKnockdown of YULINK during the arterial-venous developmental stage of zebrafish embryos led to the defective venous formation and abnormal vascular plexus formation. Knockdown of YULINK in HUVECs impaired their ability to undergo cell migration and differentiation into a capillary-like tube formation. In addition, the phosphorylated EPHB4 was decreased in YULINK knockdown HUVECs. Yeast two-hybrid, FLIM-FRET, immunoprecipitation, as well as imaging technologies showed that YULINK colocalized with endosome related proteins (EPS15, RAB33B or TICAM2) and markers (Clathrin and RHOB). VEGF-induced VEGFR2 internalization was also compromised in YULINK knockdown HUVECs, demonstrating to the involvement of YULINK.ConclusionThis study suggests that YULINK regulates vasculogenesis, possibly through endocytosis in zebrafish and HUVECs.

Nanoparticle facilitated stacked-dsRNA improves suppression of the Lepidoperan pest Chilo suppresallis

BackgroundIn recent years, gene knockdown technology using double-stranded RNA (dsRNA) has been widely used as an environment-friendly pest control strategy, but its instability and limited cellular uptake have limited its overall effect. Studies have shown that the efficiency of single dsRNA can be improved by using various nanomaterials. However, the effect of stacked-dsRNA wrapped by nanomaterial on pests remains unclear. In the present study, both CYP15C1 and C-factor genes were cloned from the midgut of C. suppressalis, and the transcript of C-factor is most highly expressed in heads. Feeding a dsCYP15C1 or dsC-factor - nanomaterial mixture can downregulate the gene expression and significantly increase larval mortality. More importantly, feeding the stacked-dsRNA wrapped by nanomaterial can significantly increase the mortality of C. suppressalis, compared with feeding dsCYP15C1 or dsC-factor - nanomaterial mixture alone. These results showed that CYP15C1 and C-factor could be potential targets for an effective management of C. suppressalis, and we developed a nanoparticle-facilitated stacked-dsRNA strategy in the control of C. suppresallis. Our research provides a theoretical basis for gene function analysis and field pest control, and will promote the application of RNAi technology in the stacked style of pest control.

Type III-A CRISPR systems as a versatile gene knockdown technology.

CRISPR-Cas systems are functionally diverse prokaryotic antiviral defense systems, which encompass six distinct types (I-VI) that each encode different effector Cas nucleases with distinct nucleic acid cleavage specificities. By harnessing the unique attributes of the various CRISPR-Cas systems, a range of innovative CRISPR-based DNA and RNA targeting tools and technologies have been developed. Here, we exploit the ability of type III-A CRISPR-Cas systems to carry out RNA-guided and sequence-specific target RNA cleavage for establishment of research tools for post-transcriptional control of gene expression. Type III-A systems from three bacterial species (L. lactis, S. epidermidis, and S. thermophilus) were each expressed on a single plasmid in E. coli, and the efficiency and specificity of gene knockdown was assessed by northern blot and transcriptomic analysis. We show that engineered type III-A modules can be programmed using tailored CRISPR RNAs to efficiently knock down gene expression of both coding and noncoding RNAs in vivo. Moreover, simultaneous degradation of multiple cellular mRNA transcripts can be directed by utilizing a CRISPR array expressing corresponding gene-targeting crRNAs. Our results demonstrate the utility of distinct type III-A modules to serve as specific and effective gene knockdown platforms in heterologous cells. This transcriptome engineering technology has the potential to be further refined and exploited for key applications including gene discovery and gene pathway analyses in additional prokaryotic and perhaps eukaryotic cells and organisms.

Role of SVEP1 in Stroma-Dependent Hematopoiesis In vitro.

Study of the microenvironment that supports hematopoietic stem cell (HSC) development in vivo is very difficult involving small numbers of interacting cells which are usually not well defined. While much is known about HSC niches located within the bone marrow in terms of contributing cell types and signalling molecules, very little is known about equivalent niches within spleen. Extramedullary hematopoiesis in spleen contributes myeloid cells important in the mobilisation of an immune response. As a result, it is important to develop in vitro models to identify the cells which constitute HSC niches in spleen and to identify the regulatory molecules supporting myeloid cell development. Studies described here document a model system to study the maintenance and differentiation of HSC by splenic stromal cells in vitro. The splenic stromal lines 5G3 and 3B5 differ in hematopoietic support capacity. SVEP1 and IGF2 are molecules of interest specifically expressed by 5G3 stroma. Gene knockdown technology using shRNA plasmids has been used to reduce gene expression in 5G3 and to determine specific effects on myeloid cell development following co-culture with overlaid hematopoietic progenitors in vitro. Knockdown of Svep1 gave specific inhibition of a dendritic cell (DC) population described previously in spleen (L-DC). Knockdown of Igf2 resulted in loss of production of a minor subset of conventional (c) DC. SVEP1 is now considered a marker of mesenchymal stromal cells with osteogenic differentiative capacity reflective of perivascular stromal cells. The power of this in vitro model is evidenced by the fact that it has been used to define SVEP1 as a specific adhesion molecule that regulates the hematopoietic process dependent on stromal niche interaction. The identification of stromal cells and molecules that contribute to the hematopoietic process in spleen, brings us closer to the realm of therapeutically regulating hematopoiesis in vivo, and to inhibiting niches which support cancer stem cells.

Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing

ABSTRACT CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific response to invading nucleic acids. Therefore, these systems can be harnessed for gene knockdown technologies even in hyperthermophilic archaea to study essential genes. We show here the broader usability of this posttranscriptional silencing technology by expanding the application to further essential genes and systematically analysing and comparing silencing thresholds and escape mutants. Synthetic guide RNAs expressed from miniCRISPR cassettes were used to silence genes involved in cell division (cdvA), transcription (rpo8), and RNA metabolism (smAP2) of the two crenarchaeal model organisms Saccharolobus solfataricus and Sulfolobus acidocaldarius. Results were systematically analysed together with those obtained from earlier experiments of cell wall biogenesis (slaB) and translation (aif5A). Comparison of over 100 individual transformants revealed gene-specific silencing maxima ranging between 40 and 75%, which induced specific knockdown phenotypes leading to growth retardation. Exceedance of this threshold by strong miniCRISPR constructs was not tolerated and led to specific mutation of the silencing miniCRISPR array and phenotypical reversion of cultures. In two thirds of sequenced reverted cultures, the targeting spacers were found to be precisely excised from the miniCRISPR array, indicating a still hypothetical, but highly active recombination system acting on the dynamics of CRISPR spacer arrays. Our results indicate that CRISPR type III – based silencing is a broadly applicable tool to study in vivo functions of essential genes in Sulfolobales which underlies a specific mechanism to avoid malignant silencing overdose.

Screening Genes Promoting Exit from Naive Pluripotency Based on Genome-Scale CRISPR-Cas9 Knockout.

Two of the main problems of stem cell and regenerative medicine are the exit of pluripotency and differentiation to functional cells or tissues. The answer to these two problems holds great value in the clinical translation of stem cell as well as regenerative medicine research. Although piling researches have revealed the truth about pluripotency maintenance, the mechanisms underlying pluripotent cell self-renewal, proliferation, and differentiation into specific cell lineages or tissues are yet to be defined. To this end, we took full advantage of a novel technology, namely, the genome-scale CRISPR-Cas9 knockout (GeCKO). As an effective way of introducing targeted loss-of-function mutations at specific sites in the genome, GeCKO is able to screen in an unbiased manner for key genes that promote exit from pluripotency in mouse embryonic stem cells (mESCs) for the first time. In this study, we successfully established a model based on GeCKO to screen the key genes in pluripotency withdrawal. Our strategies included lentiviral package and infection technology, lenti-Cas9 gene knockout technology, shRNA gene knockdown technology, next-generation sequencing, model-based analysis of genome-scale CRISPR-Cas9 knockout (MAGeCK analysis), GO analysis, and other methods. Our findings provide a novel approach for large-scale screening of genes involved in pluripotency exit and offer an entry point for cell fate regulation research.

CRISPR-mediated gene silencing reveals involvement of the archaeal S-layer in cell division and virus infection

The S-layer is a proteinaceous surface lattice found in the cell envelope of bacteria and archaea. In most archaea, a glycosylated S-layer constitutes the sole cell wall and there is evidence that it contributes to cell shape maintenance and stress resilience. Here we use a gene-knockdown technology based on an endogenous CRISPR type III complex to gradually silence slaB, which encodes the S-layer membrane anchor in the hyperthermophilic archaeon Sulfolobus solfataricus. Silenced cells exhibit a reduced or peeled-off S-layer lattice, cell shape alterations and decreased surface glycosylation. These cells barely propagate but increase in diameter and DNA content, indicating impaired cell division; their phenotypes can be rescued through genetic complementation. Furthermore, S-layer depleted cells are less susceptible to infection with the virus SSV1. Our study highlights the usefulness of the CRISPR type III system for gene silencing in archaea, and supports that an intact S-layer is important for cell division and virus susceptibility.

PD-1 silencing impairs the anti-tumor function of chimeric antigen receptor modified T cells by inhibiting proliferation activity

BackgroundBlocking programmed death-1 (PD-1) is considered to be a promising strategy to improve T cell function, and this is being explored in many ongoing clinical trials. In fact, our knowledge about PD-1 is primarily based on the results of short-term experiments or observations, but how long-lasting PD-1 blockade can affect T cell function remains unclear.MethodsWe planned to use shRNA-based gene knockdown technology to mimic long-lasting PD-1 blockade. We constructed PD-1 steadily blocked chimeric antigen receptor modified T (CAR-T) cells, and with these cells we can clearly study the effects of PD-1 knockdown on T cell function. The anti-tumor function, proliferation ability and differentiation status of PD-1 silenced CAR-T cells were studied by in vitro and animal experiments.ResultsAccording to short-term in vitro results, it was reconfirmed that the resistance to programmed death-ligand 1 (PD-L1)-mediated immunosuppression could be enhanced by PD-1 blockade. However, better anti-tumor function was not presented by PD-1 blocked CAR-T cells in vitro or in vivo experiments. It was found that PD-1 knockdownmight impair the anti-tumor potential of CAR-T cells because it inhibited T cells’ proliferation activity. In addition, we observed that PD-1 blockade would accelerate T cells’ early differentiation and prevent effector T cells from differentiating into effect memory T cells, and this might be the reason for the limited proliferation of PD-1 silenced CAR-T cells.ConclusionThese results suggest that PD-1 might play an important role in maintaining the proper proliferation and differentiation of T cells, and PD-1 silencing would impair T cells’ anti-tumor function by inhibiting their proliferation activity.

ABIN-1 Negatively Regulates μ-Opioid Receptor Function.

The μ-opioid receptor (MOR) is a Gi/o protein-coupled receptor that mediates analgesic, euphoric, and reward effects. Using a bacterial two-hybrid screen, we reported that the carboxyl tail of the rat MOR associates with A20-binding inhibitor of nuclear factor κB (ABIN-1). This interaction was confirmed by direct protein-protein binding and coimmunoprecipitation of MOR and ABIN-1 proteins in cell lysates. Saturation binding studies showed that ABIN-1 had no effect on MOR binding. However, the interaction of ABIN-1 and MOR inhibited the activation of G proteins induced by DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin). MOR phosphorylation, ubiquitination, and internalization induced by DAMGO were decreased in Chinese hamster ovary cells that coexpressed MOR and ABIN-1. The suppression of forskolin-stimulated adenylyl cyclase by DAMGO was also inhibited by the interaction of ABIN-1 with MOR. In addition, extracellular signal-regulated kinase activation was also negatively regulated by overexpression of ABIN-1. These data suggest that ABIN-1 is a negative coregulator of MOR activation, phosphorylation, and internalization in vitro. ABIN-1 also inhibited morphine-induced hyperlocomotion in zebrafish larvae (AB strain). By utilization of an antisense morpholino oligonucleotide (MO) gene knockdown technology, the ABIN-1 MO-injected zebrafish larvae showed a significant increase (approximately 60%) in distance moved compared with control MO-injected larvae after acute morphine treatment (P < 0.01). Taken together, ABIN-1 negatively regulates MOR function in vitro and in vivo.

Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9

The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.

Efficient Gene Knockdowns in Mouse Embryonic Stem Cells Using MicroRNA-Based shRNAs.

RNA interference (RNAi) is a powerful gene knockdown technology that has been applied for functional genetic loss-of-function studies in many model eukaryotic systems, including embryonic stem cells (ESCs). Application of RNAi in ESCs allows for dissection of mechanisms by which ESCs self-renew and maintain pluripotency and also for specifying particular cell types needed for cell replacement therapies. Potent RNAi response can be induced by expression of a microRNA-embedded short-hairpin RNA (shRNAmir) cassette that is integrated in the genome by virus infection or site-specific recombination at a defined locus. In this chapter, I will provide detailed protocols to perform shRNAmir-mediated RNAi studies in mouse ESCs using retrovirus infection and loxP site-directed recombination for efficient constitutive and inducible gene knockdown, respectively.

Computing Molecular Devices in L.major through Transcriptome Analysis: Structured Simulation Approach.

In the modern era of post genomics and transcriptomics, non-coding RNAs and non-coding regions of many RNAs are a big puzzle when we try deciphering their role in specific gene function. Gene function assessment is a main task wherein high throughput technologies provide an impressive body of data that enables the design of hypotheses linking genes to phenotypes. Gene knockdown technologies and RNA-dependent gene silencing are the most frequent approaches to assess the role of key effectors in a particular scenario. Ribozymes are effective modulators of gene expression because of their simple structure, site-specific cleavage activity, and catalytic potential. In our study, after an extensive transcriptomic search of Leishmania major transcriptome we found a Putative ATP dependent DNA helicase (Lmjf_09_0590) 3’ UTR which has a structural signature similar to well-known HDV hammerhead ribozyme, even though they have variable sequence motifs. Henceforth, to determine their structural stability and sustainability we analyzed our predicted structural model of this 3’UTR with a 30ns MD simulation, further confirmed with 100ns MD simulation in presence of 5mM MgCl2 ionic environment. In this environment, structural stability was significantly improved by bonded interactions between a RNA backbone and Mg2+ ions. These predictions were further validated in silico using RNA normal mode analysis and anisotropic network modelling (ANM) studies. The study may be significantly imparted to know the functional importance of many such 3’UTRs to predict their role in a mechanistic manner.

Abstract 1106: Genome-wide CRISPR-Cas9 screens reveal candidate therapeutic targets and tumor suppressor genes for human glioma

Abstract Glioblastoma multiforme (GBM) is the most aggressive and common form of brain cancer in adults. There are currently no effective therapies for GBM. Even with standard of care treatments, such as surgery, radiation, and chemotherapy, ∼90% of adult patients die within 2 years of diagnosis. Our inability to develop new more effective therapies may arise from pre-clinical models that inadequately predict therapeutic window and the fact that many new GBM drugs are “hand-me-downs” from other cancers, not specifically developed for treating brain tumors. To identify patient-tailored drug targets for GBM, our group has performed a series of functional genetic screens in patient derived GBM stem-like cells (GSCs) and also non-transformed human neural stem cells (NSCs). GSCs retain tumor-initiating potential and tumor-specific genetic and epigenetic signatures, even during extended outgrowth in serum-free culture. NSCs represent non-transformed candidate cell of origin controls, which share similar gene expression signatures and identical in vitro growth conditions. Using these systems along with RNAi or CRISPR/Cas9 platforms, we have identified multiple molecular vulnerabilities specific to GSCs, which appear to be largely driven by oncogenic transformation, in processes ranging from kinetochore regulation to 3′ pre-mRNA splice site recognition. At this meeting, we present our latest findings from genome-wide CRISPR-Cas9 gene knockout screens in multiple GSC and NSC isolates. These include validation studies of patient tumor-lethal genes using gene knockout rather than gene knockdown technology. In addition, we will present validation studies of gene products growth limiting for NSC expansion/self-renewal from these screens, a subset of which are candidate tumor suppressors for glioma. Strengths and weaknesses of using gene editing technology and GSC isolates for identification of therapeutic targets will be discussed. Citation Format: Yu Ding, Chad Toledo, Pia Hoellerbauer, Ryan Basom, Emily Girad, Eunjee Lee, Philip Corrin, Qi Lin, Xiao-Nan Li, Do-Hyun Nam, Jeongwu Lee, Jun Zhu, Steven Pollard, Jeffery Delrow, Jim Olson, Patrick J. Paddison. Genome-wide CRISPR-Cas9 screens reveal candidate therapeutic targets and tumor suppressor genes for human glioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1106. doi:10.1158/1538-7445.AM2015-1106

Study of mRNA turnover never decays.

Study of mRNA turnover never decays.

Seed sequence-matched controls reveal limitations of small interfering RNA knockdown in functional and structural studies of hepatitis C virus NS5A-MOBKL1B interaction.

Hepatitis C virus (HCV) is a widespread human pathogen causing liver cirrhosis and cancer. Similar to the case for other viruses, HCV depends on host and viral factors to complete its life cycle. We used proteomic and yeast two-hybrid approaches to elucidate host factors involved in HCV nonstructural protein NS5A function and found that MOBKL1B interacts with NS5A. Initial experiments with small interfering RNA (siRNA) knockdown suggesting a role in HCV replication led us to examine the interaction using biochemical and structural approaches. As revealed by a cocrystal structure of a core MOBKL1B-NS5A peptide complex at 1.95 Å, NS5A binds to a hydrophobic patch on the MOBKL1B surface. Biosensor binding assays identified a highly conserved, 18-amino-acid binding site in domain II of NS5A, which encompasses residues implicated in cyclophilin A (CypA)-dependent HCV RNA replication. However, a CypA-independent HCV variant had reduced replication in MOBKL1B knockdown cells, even though its NS5A does not interact with MOBKL1B. These discordant results prompted more extensive studies of MOBKL1B gene knockdowns, which included additional siRNAs and specifically matched seed sequence siRNA controls. We found that reduced virus replication after treating cells with MOBKL1B siRNA was actually due to off-target inhibition, which indicated that the initial finding of virus replication dependence on the MOBKL1B-NS5A interaction was incorrect. Ultimately, using several approaches, we found no relationship of the MOBKL1B-NS5A interaction to virus replication. These findings collectively serve as a reminder to investigators and scientific reviewers of the pervasive impact of siRNA off-target effects on interpretation of biological data. Our study illustrates an underappreciated shortcoming of siRNA gene knockdown technology. We initially identified a cellular protein, MOBKL1B, as a binding partner with the NS5A protein of hepatitis C virus (HCV). MOBKL1B siRNA, but not irrelevant RNA, treatment was associated with both reduced virus replication and the absence of MOBKL1B. Believing that HCV replication depended on the MOBKL1B-NS5A interaction, we carried out structural and biochemical analyses. Unexpectedly, an HCV variant lacking the MOBKL1B-NS5A interaction could not replicate after cells were treated with MOBKL1B siRNA. By repeating the MOBKL1B siRNA knockdowns and including seed sequence-matched siRNA instead of irrelevant siRNA as a control, we found that the MOBKL1B siRNAs utilized had off-target inhibitory effects on virus replication. Collectively, our results suggest that stricter controls must be utilized in all RNA interference (RNAi)-mediated gene knockdown experiments to ensure sound conclusions and a reliable scientific knowledge database.

Functional genetics for all: engineered nucleases, CRISPR and the gene editing revolution.

Developmental biology, as all experimental science, is empowered by technological advances. The availability of genetic tools in some species - designated as model organisms - has driven their use as major platforms for understanding development, physiology and behavior. Extending these tools to a wider range of species determines whether (and how) we can experimentally approach developmental diversity and evolution. During the last two decades, comparative developmental biology (evo-devo) was marked by the introduction of gene knockdown and deep sequencing technologies that are applicable to a wide range of species. These approaches allowed us to test the developmental role of specific genes in diverse species, to study biological processes that are not accessible in established models and, in some cases, to conduct genome-wide screens that overcome the limitations of the candidate gene approach. The recent discovery of CRISPR/Cas as a means of precise alterations into the genome promises to revolutionize developmental genetics. In this review we describe the development of gene editing tools, from zinc-finger nucleases to TALENs and CRISPR, and examine their application in gene targeting, their limitations and the opportunities they present for evo-devo. We outline their use in gene knock-out and knock-in approaches, and in manipulating gene functions by directing molecular effectors to specific sites in the genome. The ease-of-use and efficiency of CRISPR in diverse species provide an opportunity to close the technology gap that exists between established model organisms and emerging genetically-tractable species.Electronic supplementary materialThe online version of this article (doi:10.1186/2041-9139-5-43) contains supplementary material, which is available to authorized users.

A morpholino‐based screen to identify novel genes involved in craniofacial morphogenesis

The regulatory mechanisms underpinning facial development are conserved between diverse species. Therefore, results from model systems provide insight into the genetic causes of human craniofacial defects. Previously, we generated a comprehensive dataset examining gene expression during development and fusion of the mouse facial prominences. Here, we used this resource to identify genes that have dynamic expression patterns in the facial prominences, but for which only limited information exists concerning developmental function. This set of ∼80 genes was used for a high-throughput functional analysis in the zebrafish system using Morpholino gene knockdown technology. This screen revealed three classes of cranial cartilage phenotypes depending upon whether knockdown of the gene affected the neurocranium, viscerocranium, or both. The targeted genes that produced consistent phenotypes encoded proteins linked to transcription (meis1, meis2a, tshz2, vgll4l), signaling (pkdcc, vlk, macc1, wu:fb16h09), and extracellular matrix function (smoc2). The majority of these phenotypes were not altered by reduction of p53 levels, demonstrating that both p53-dependent and -independent mechanisms were involved in the craniofacial abnormalities. This Morpholino-based screen highlights new genes involved in development of the zebrafish craniofacial skeleton with wider relevance to formation of the face in other species, particularly mouse and human.

Molecular analysis of central feeding regulation by neuropeptide Y (NPY) neurons with NPY receptor small interfering RNAs (siRNAs)

Hypothalamic neuropeptides play important roles in central feeding behavior. Among them, neuropeptide Y (NPY) has the strongest orexigenic action. It is synthesized in NPY-expressing neurons in the arcuate nucleus (ARC), which projects to other nuclei, mainly to the paraventricular nucleus (PVN). PVN, which possesses NPY-Y1, -Y2 and -Y4, -Y5 receptors, is considered as feeding center for central feeding behavior. Herein I review recent results on feeding behavior obtained by gene knockdown technologies. The small interfering RNA (siRNA) plasmid-based vectors, which drive transcription of siRNA by U6 RNA polymerase III promoter to produce knockdown of the NPY and its receptor (Y1, Y2, Y4 and Y5) genes, were stereotaxically injected into mouse ARC and PVN. Feeding behaviors were measured for 6days after siRNA vector injection. NPY and its receptor mRNA levels were decreased, which were measured by RT-PCR and in situ hybridization, and simultaneous decrease in their proteins was also detected in separate nuclei by immunohistochemistry. In the NPY system, decrease in NPY, Y1 and Y5 expressions in specialized nuclei diminished central feeding behavior, whereas decrease in Y2 or Y4 expression in both ARC or PVN did not affect feeding behavior. Thus, specialized change in expressions of NPY and its receptors (especially Y1 and Y5) are important for regulation of endogenous feeding behavior in central regulation. Further analysis of NPY receptors may provide better understanding of feeding behavior and of potential therapeutic targets.