Embryo Injection Research Articles

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in <em>Nasonia vitripennis</em>

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

One-Step Homology Mediated CRISPR-Cas Editing in Zygotes for Generating Genome Edited Cattle.

Selective breeding and genetic modification have been the cornerstone of animal agriculture. However, the current strategy of breeding animals over multiple generations to introgress novel alleles is not practical in addressing global challenges such as climate change, pandemics, and the predicted need to feed a population of 9 billion by 2050. Consequently, genome editing in zygotes to allow for seamless introgression of novel alleles is required, especially in cattle with long generation intervals. We report for the first time the use of CRISPR-Cas genome editors to introduce novel PRNP allelic variants that have been shown to provide resilience towards human prion pandemics. From one round of embryo injections, we have established six pregnancies and birth of seven edited offspring, with two founders showing >90% targeted homology-directed repair modifications. This study lays out the framework for in vitro optimization, unbiased deep-sequencing to identify editing outcomes, and generation of high frequency homology-directed repair–edited calves.

Status of racial disparities between black and white women undergoing assisted reproductive technology in the US

BackgroundNumerous studies have demonstrated substantial differences in assisted reproductive technology outcomes between black non-Hispanic and white non-Hispanic women. We sought to determine if disparities in assisted reproductive technology outcomes between cycles from black non-Hispanic and white non-Hispanic women have changed and to identify factors that may have influenced change and determine racial differences in cumulative live birth rates.MethodsThis is a retrospective cohort study of the SARTCORS database outcomes for 2014–2016 compared with those previously reported in 2004–2006 and 1999/2000. Patient demographics, etiology of infertility, and cycle outcomes were compared between black non-hispanic and white non-hispanic patients. Categorical values were compared using Chi-squared testing. Continuous variables were compared using t-test. Multiple logistic regression was used to assess confounders.ResultsWe analyzed 122,721 autologous, fresh, non-donor embryo cycles from 2014 to 2016 of which 13,717 cycles from black and 109,004 cycles from white women. The proportion of cycles from black women increased from 6.5 to 8.4%. Cycles from black women were almost 3 times more likely to have tubal and/or uterine factor and body mass index ≥30 kg/m2. Multivariate logistic regression demonstrated that black women had a lower live birth rate (OR 0.71;P < 0.001) and a lower cumulative live birth rate for their initial cycle (OR 0.64; P < 0.001) independent of age, parity, body mass index, etiology of infertility, ovarian reserve, cycle cancellation, past spontaneous abortions, use of intra-cytoplasmic sperm injection or number of embryos transferred. A lower proportion of cycles in black women were represented among non-mandated states (P < 0.001) and cycles in black women were associated with higher clinical live birth rates in mandated states (P = 0.006).ConclusionsDisparities in assisted reproductive technology outcomes in the US have persisted for black women over the last 15 years. Limited access to state mandated insurance may be contributory. Race has continued to be an independent prognostic factor for live birth and cumulative live birth rate from assisted reproductive technology in the US.

Cas9-Mediated Gene-Editing in the Black-Legged Tick, &lt;i&gt;Ixodes Scapularis&lt;/i&gt;, by Embryo Injection and ReMOT Control

Despite their capacity to acquire and pass on an array of debilitating pathogens, research on ticks has lagged behind other arthropod vectors, such as mosquitoes, largely because of a lack of genetic and molecular tools. CRISPR-Cas9 is transforming non-model organism research; however, successful  gene editing has not yet been reported in ticks. Technical challenges for injecting tick embryos to attempt gene editing have further slowed research progress. Herein we report a successful tick embryo injection protocol for the black-legged tick, Ixodes scapularis, the first for any chelicerate species, and the use of this protocol for genome editing with CRISPR-Cas9. We also demonstrated for the first time that the ReMOT Control technique can be successfully used to generate genome mutations outside Insecta. Our results provide innovative tools to the tick research community that are essential for advancing our understanding of the molecular mechanisms governing pathogen transmission by tick vectors as well as for understanding the underlying biology of host-vector-pathogen interactions.

Embryo location in the uterus during embryo transfer: An in vitro simulation.

ObjectiveTo evaluate the location of transferred embryos under various parameters during embryo transfer in in vitro fertilization (IVF) by applying an in vitro experimental model for embryo transfer (ET).MethodsMock ET simulations were conducted with a laboratory model of the uterine cavity. The transfer catheter was loaded with a sequence of air and liquid volumes, including development-arrested embryos donated by patients. The transfer procedure was recorded using a digital video camera. An orthogonal design, including three independent variables (uterine orientation, distance of the catheter tip to the fundus, and injection speed) and one dependent variable (final embryo position), was applied.ResultsThe uterine cavity was divided into six regions. The distribution of the transferred matter within the uterine cavity varied according to the uterine orientation. Medium speed-injected embryos were mostly found in the static region while fast- and slow-speed injected embryos were mostly found in the fundal region and the cervical-left region, respectively. The possibility of embryo separation from the air bubble increased from 11.1% in slow injection cases to 29.6% and 48.1% in medium and fast injection cases, respectively.ConclusionThe experimental model provides a new method for investigating ET procedures. Fast injection of embryos into a retroverted uterus may be more likely to result in embryo separation from the air bubble.

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.

GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos.

Genome editing holds great potential for correcting pathogenic mutations. We developed a method called GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus could detect potential off-target variants with high sensitivity. Notably, the GOTI method was designed to detect potential off-target variants of any genome editing tools by the combination of experimental and computational approaches, which is critical for accurate evaluation of the safety of genome editing tools. Here we provide a detailed protocol for GOTI, including mice mating, two-cell embryo injection, embryonic day 14.5 embryo digestion, fluorescence-activated cell sorting, whole-genome sequencing and data analysis. To enhance the utility of GOTI, we also include a computational workflow called GOTI-seq (https://github.com/sydaileen/GOTI-seq) for the sequencing data analysis, which can generate the final genome-wide off-target variants from raw sequencing data directly. The protocol typically takes 20 d from the mice mating to sequencing and 7 d for sequencing data analysis.

Mthfd2 Modulates Mitochondrial Function and DNA Repair to Maintain the Pluripotency of Mouse Stem Cells.

SummaryThe pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells.

Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects.

Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5'-CC-3' motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.

Over-expression of human PP5 gene in mice induces corneal hyperplasia and leads to ocular surface squamous neoplasia

Protein phosphatase 5 (PP5) plays an important role in cell proliferation, differentiation, and development. Transgenic PP5 mice (Tg-hPP5 mice) overexpressing human PP5 gene were successfully generated by embryo injection. Tg-hPP5 mice spontaneously developed corneal hyperplasia and ocular surface squamous neoplasia (OSSN). To investigate the mechanism behind PP5-induced corneal hyperplasia, we performed immunohistochemistry, quantitative real-time PCR, and Western Blotting analyses on the corneas of Tg-hPP5 mice at 2 months and 9 months of age. We provide the first demonstration that Tg-hPP5 mice develop corneal hyperplasia at 9-months of age demonstrated via histological analysis and in vitro co-transfection investigation. We also present data that the expression of p53 is significantly reduced while the expression of FGF-7 is significantly increased in Tg-hPP5 mice with corneal hyperplasia. Co-transfection of PP5, p53, and FGF-7-promoter-driven luciferase revealed that PP5 promotes while p53 inhibits FGF-7 expression, which indicates PP5 overexpression inhibits p53 phosphorylation, thereby reducing its tumor suppressor function and increasing FGF-7 expression. In conclusion, PP5 plays a pivotal role in corneal hyperplasia development and its downregulation is a potential target for corneal hyperplasia and OSSN treatment.

Study on Simulation of Injection Process and Optimization of Process Parameters of Polypropylene Bottle Embryo

The purpose of this paper is to obtain the best process parameters of polypropylene bottle injection molding. This paper obtained the gating system of polypropylene bottle embryo by using MOLDFLOW according to the structural characteristics of polypropylene bottle embryo, and optimized the process parameters by using taguchi method within the reasonable range of process parameters. In this paper, six parameters about time, pressure and temperature were selected as factors for the mold flow analysis of polypropylene bottle embryo injection molding, and there were four levels in every factor. The volume shrinkage, sink index and residual stress in the first and second directions in the cavity were selected as evaluation indexes. In order to reduce evaluation indexes, the signal to noise ratio of each factor and each level were calculated, and the adjustable factors were adjusted to obtain the minimum evaluation indexes and optimal combination of process parameters. The optimum process parameters such as melt temperature 200°C, molding temperature 20°C, injection pressure 50Mpa, injection time 2.1s,dwell pressure 44Mpa, dwell time 40s were gained.

Role of the insulin receptor‐related receptor in Xenopus laevis development

The orphan insulin receptor‐related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin‐like growth factor receptor (IGF‐IR) can be activated by mildly alkaline extracellular medium. Unlike ubiquitously expressed IR and IGF‐IR, IRR is found in specific sets of cells in only some tissues, most of them being exposed to extracorporal liquids of extreme pH. In the evolution, IRR is highly conserved since its divergence from the insulin and insulin‐like growth factor receptors in amphibia. IR and IGF‐IR signaling are important in growth and development in zebrafish or frog embryogenesis. In contrast, the role of IRR activation during embryogenesis is unknown although Xenopus embryo have strong IRR expression. To address this, we examined the function of the IRR during Xenopus laevis development by morpholino‐mediated selective knockdown of IRR. We demonstrated that inhibition of IRR expression in Xenopus laevis leads to delayed development, but this phenotype can be restored by incubation of embryos in alkaline medium. Also, using RNA‐seq of total RNA we showed that IRR inhibition dramatically changed the embryo transcriptome, and partially restored after alkali treatment. We have identified several hundred genes (eomes, frzb, pax6 etc.) whose expression changes after IRR knockdown and restores by alkali exposure. Our results clearly demonstrate that IRR plays previously unidentified important role in frog embryogenesis and growth.Support or Funding InformationThis work was financially supported by the Russian Foundation for Basic Research (grants No.19‐04‐00815, 17‐00‐00486)To understand which role IRR could play in the embryonic development, we downregulated it by injecting anti‐sense morpholino oligonucleotide to IRR mRNA (MO1 xIRR) into the two‐cells embryos (Fig. 1A). As a result, we observed significant retardation of development of these embryos comparing to their siblings injected by the control MO (Fig. 1B,C). As IRR in mouse is known as a receptor which activity depends on pH, we decided to verify if pH different from the neutral one could interfere with the effects observed in embryos with downregulated pH. To this end, we incubated embryos injected with IRR MO and by the control one in solutions with pH 5.5, 7.2 and 8.4.Whereas clear retardation of development was seen in the MO1 xIRR injected embryos incubated in pH 5.2 and 7.2 (Fig, 1B,C,D,E). Surprisingly, the embryos incubated at pH 8.4 developed approximately synchronously with the control ones (Fig. 1F,G). In other words, unexpectedly alkaline pH “rescues” embryos with downregulated IRR from retardation of development.We represented in table 1 top of 15 up‐regulated and 15 down‐regulated genes after MO injection in embryos at pH 7.2, which included several key transcription factors such as eomes, pou5f3.2 (or oct25), hhex and pax6. Also, we see that genes with the largest modified expression are actin or myosin coding genes.Figure 1 gene Fold change MO xIRR 7.2/control 7.2 Fold change MO xIRR 8.4/MO xIRR 7.2 mylpf.L 0,15 2,95 myosin light chain, phosphorylatable, fast skeletal muscle S homeolog(mylpf.S) act3.L 0,15 1,89 actin, alpha skeletal muscle MGC64484 0,15 1,89 Actin, alpha cardiac muscle 1 act2 0,20 2,32 actin, alpha sarcomeric/cardiac myl1.S 0,25 1,93 myosin light chain 1 S homeolog MGC53823 0,25 1,99 Actin, alpha cardiac muscle 2‐like col2a1.L 0,26 2,07 collagen, type II, alpha 1 L homeolog des.1.L 0,26 2,11 desmin, gene 1 L homeolog fbxl22.S 0,30 1,94 F‐box and leucine‐rich repeat protein 22 S homeolog tnnc2.L 0,30 1,68 troponin C type 2 (fast) L homeolog pax6.S 0,30 2,43 paired box 6 S homeolog des.1.S 0,30 1,95 desmin, gene 1 S homeolog smyd1.L 0,31 2,21 SET and MYND domain containing 1 L homeolog tnnt3.L 0,32 2,26 troponin T3, fast skeletal type L homeolog nr2f5.S 0,32 1,87 nuclear receptor subfamily 2, group F, member 5 S homeolog dctn2.L 1,92 0,58 dynactin subunit 2 L homeolog pou5f3.2.L 1,93 0,51 POU class 5 homeobox 3, gene 2 L homeolog ywhag.L 1,93 0,50 tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein, gamma L homeolog ppp2r2b.L 1,93 0,52 protein phosphatase 2 regulatory subunit B, beta L homeolog chm.L 1,97 0,56 choroideremia (Rab escort protein 1) L homeolog hhex.L 1,97 0,62 hematopoietically expressed homeobox L homeolog slc22a15.2.S 1,97 0,63 solute carrier family 22, member 15, gene 2 S homeolog gramd1c.L 1,97 0,70 GRAM domain containing 1C L homeolog ccna1.L 1,99 0,48 cyclin A1 L homeolog neu1.L 2,00 0,56 neuraminidase 1 (lysosomal sialidase) L homeolog cdk5r2.S 2,03 0,57 cyclin‐dependent kinase 5, regulatory subunit 2 (p39) S homeolog cer1.S 2,11 0,54 cerberus 1, DAN family BMP antagonist S homeolog xpo6.S 2,19 0,67 exportin 6 S homeolog eomes.S 2,42 0,36 eomesodermin S homeolog frzb.S 2,71 0,39 frizzled‐related protein S homeolog

CRISPR-Cas9-Based Genome Editing in the Silverleaf Whitefly (Bemisia tabaci).

Bemisia tabaci cryptic species Middle East-Asia Minor I (MEAM1) is a serious agricultural polyphagous insect pest and vector of numerous plant viruses, causing major worldwide economic losses. B. tabaci control is limited by lack of robust gene editing tools. Gene editing is difficult in B. tabaci due to small embryos that are technically challenging to inject and which have high mortality post injection. We developed a CRISPR-Cas9 gene editing protocol based on injection of vitellogenic adult females rather than embryos ("ReMOT Control"). We identified an ovary-targeting peptide ligand ("BtKV") that, when fused to Cas9 and injected into adult females, transduced the ribonucleoprotein complex to the germline, resulting in efficient, heritable editing of the offspring genome. In contrast to embryo injection, adult injection is easy and does not require specialized equipment. Development of easy-to-use gene editing protocols for B. tabaci will allow researchers to apply the power of reverse genetic approaches to this species and will lead to novel control methods for this devastating pest insect.

Tyrosine pre-transfer RNA fragments are linked to p53-dependent neuronal cell death via PKM2

Fragments of transfer RNA (tRNA), derived either from pre-tRNA or mature tRNA, have been discovered to play an essential role in the pathogenesis of various disorders such as neurodegenerative disease. CLP1 is an RNA kinase involved in tRNA biogenesis, and mutations in its encoding gene are responsible for pontocerebellar hypoplasia type-10. Mutation of the CLP1 gene results in the accumulation of tRNA fragments of several different kinds. These tRNA fragments are expected to be associated with the disease pathogenesis. However, it is still unclear which of the tRNA fragments arising from the CLP1 gene mutation has the greatest impact on the onset of neuronal disease. We found that 5′ tRNA fragments derived from tyrosine pre-tRNA (5′ Tyr-tRF) caused p53-dependent neuronal cell death predominantly more than other types of tRNA fragment. We also showed that 5′ Tyr-tRF bound directly to pyruvate kinase M2 (PKM2). Injection of zebrafish embryos with PKM2 mRNA ameliorated the neuronal defects induced in zebrafish embryos by 5′ Tyr-tRF. Our findings partially uncovered a mechanistic link between 5′ Tyr-tRF and neuronal cell death that is regulated by PKM2.

Cas9-Mediated Gene-Editing in the Malaria Mosquito Anopheles stephensi by ReMOT Control.

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.

A method for CRISPR/Cas9 mutation of genes in fathead minnow (Pimephales promelas)

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing allows for the disruption or modification of genes in a multitude of model organisms. In the present study, we describe and employ the method for use in the fathead minnow (Pimephales promelas), in part, to assist in the development and validation of adverse outcome pathways (AOPs). The gene coding for an enzyme responsible for melanin production, tyrosinase (tyr), was the initial target chosen for development and assessment of the method since its disruption results in abnormal pigmentation, a phenotype obvious within 3–4 d after injection of fathead minnow embryos. Three tyrosinase-targeting guide strands were generated using the fathead minnow sequence in tandem with the CRISPOR guide strand selection tool. The strands targeted two areas: one stretch of sequence in a conserved region that demonstrated homology to EGF-like or laminin-like domains as determined by Protein Basic Local Alignment Search Tool in concert with the Conserved Domain Database, and a second area in the N-terminal region of the tyrosinase domain. To generate one cell embryos, in vitro fertilization was performed, allowing for microinjection of hundreds of developmentally-synchronized embryos with Cas9 proteins complexed to each of the three guide strands. Altered retinal pigmentation was observed in a portion of the tyr guide strand injected population within 3 d post fertilization (dpf). By 14 dpf, fish without skin and swim bladder pigmentation were observed. Among the three guide strands injected, the guide targeting the EGF/laminin-like domain was most effective in generating mutants. CRISPR greatly advances our ability to directly investigate gene function in fathead minnow, allowing for advanced approaches to AOP validation and development.

CRISPR/Cas9-mediated deletion of one carotenoid isomerooxygenase gene (EcNinaB-X1) from Exopalaemon carinicauda

During the immune defense reaction of invertebrate, a plenty of reactive oxygen species (ROS) could be induced to product. Though ROS can kill foreign invaders, the accumulation of these reactive molecules in animals will cause serious cell damage. Carotenoids could function as scavengers of oxygen radicals. In this research, cDNA and genomic DNA of one carotenoid isomerooxygenase gene (named EcNinaB-X1) were cloned from Exopalaemon carinicauda. EcNinaB-X1 gene was composed of 12 exons and 11 introns. EcNinaB-X1 knock-out (KO) prawns were produced via CRISPR/Cas9 technology and the change of their phenotypes were analyzed. Of the 400 injected one-cell stage embryos with cas9 mRNA and one sgRNA targeting the first exon of EcNinaB-X1 gene, 26 EcNinaB-X1-KO prawns were generated and the mutant rate reached 6.5% after embryo injection. The EcNinaB-X1-KO prawns had significant lower mortality than those in wild-type group when the prawns were challenged with Vibrio parahaemolyticus or Aeromonas hydrophila. In conclusion, we first demonstrate the function of the carotenoid isomerooxygenase gene in immune defense of E. carinicauda by performing directed, heritable gene mutagenesis.

Refinement of inducible gene deletion in embryos of pregnant mice.

CreERT2‐mediated gene recombination is widely applied in developmental biology research. Activation of CreERT2 is typically achieved by injection of tamoxifen in an oily vehicle into the peritoneal cavity of mid‐gestation pregnant mice. This can be technically challenging and adversely impacts welfare. Here we characterize three refinements to this technique: Pipette feeding (not gavage) of tamoxifen, ex vivo CreERT2 activation in whole embryo culture and injection of cell‐permeable TAT‐Cre into Cre‐negative cultured embryos. We demonstrate that pipette feeding of tamoxifen solution to the mother on various days of gestation reliably activates embryonic CreERT2, illustrated here using β‐Actin CreERT2, Sox2 CreERT2, T CreERT2, and Nkx1.2 CreERT2. Pipette feeding of tamoxifen induces dose‐dependent recombination of Rosa26 mTmG reporters when administered at E8.5. Activation of two neuromesodermal progenitor‐targeting Cre drivers, T CreERT2, and Nkx1.2 CreERT2, produces comparable neuroepithelial lineage tracing. Dose‐dependent CreERT2 activation can also be achieved by brief exposure to 4OH‐tamoxifen in whole embryo culture, allowing temporal control of gene deletion and eliminating the need to treat pregnant mice. Rosa26 mTmG reporter recombination can also be achieved regionally by injecting TAT‐Cre into embryonic tissues at the start of culture. This allows greater spatial control over Cre activation than can typically be achieved with endogenous CreERT2, for example by injecting TAT‐Cre on one side of the midline. We hope that our description and application of these techniques will stimulate refinement of experimental methods involving CreERT2 activation for gene deletion and lineage tracing studies. Improved temporal (ex vivo treatment) and spatial (TAT‐Cre injection) control of recombination will also allow previously intractable questions to be addressed.

Development of a film-assisted honeybee egg collection system (FECS)

Despite the huge potential of genome editing in honeybee research and breeding programs, only a few successful studies have been reported, implying the presence of several obstacles. One such obstacle is the difficulty in obtaining a large quantity of young eggs. To facilitate the overall procedures for egg collection and microinjection, we developed the film-assisted honeybee egg collection system (FECS), in which a removable transparent film is used as the platform for consecutive collection and injection of embryos. The FECS provides a significantly higher (~ 2-fold) collection efficiency and usability compared with the conventional plug-based queen rearing system while maintaining a high hatching rate. The system can be readily utilized for various studies that require a large number of honeybee eggs, including honeybee genome editing.

CRISPR/Cas9 Genome Editing Introduction and Optimization in the Non-model Insect Pyrrhocoris apterus.

The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice Mus musculus, zebrafish Danio reiro, and the fruit fly Drosophila melanogaster. However, publications describing the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species Pyrrhocoris apterus. We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method, in vivo testing of sgRNA efficiency, and G0 germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.