Efficient CRISPR-mediated genome editing can be initiated by embryonic injection but not by ovarian delivery in the beetle Tribolium castaneum.

The clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9)-mediated gene editing technology has revolutionized the study of fundamental biological questions in various insects. Diverse approaches have been developed to deliver the single-guide RNA (sgRNA) and Cas9 to the nucleus of insect embryos or oocytes to achieve gene editing, including the predominant embryonic injection methods and alternative protocols through parental ovary delivery. However, a systematic comparative study of these approaches is limited, especially within a given insect. Here, we focused on revealing the detailed differences in CRISPR/Cas9-mediated gene editing between the embryo and ovary delivery methods in the beetle Tribolium castaneum, using the cardinal and tyrosine hydroxylase (TH) as reporter genes. We demonstrated that both genes could be efficiently edited by delivering Cas9/sgRNA ribonucleoproteins to the embryos by microinjection, leading to the mutant phenotypes and indels in the target gene sites. Next, the Cas9/sgRNA complex, coupled with a nanocarrier called Branched Amphiphilic Peptide Capsules (BAPC), were delivered to the ovaries of parental females to examine the efficacy of BAPC-mediated gene editing. Although we observed that a small number of beetles' progeny targeting the cardinal exhibited the expected white-eye phenotype, unexpectedly, no target DNA indels were found following subsequent sequencing analysis. In addition, we adopted a novel approach termed "direct parental" CRISPR (DIPA-CRISPR). However, we still failed to find gene-editing events in the cardinal or TH gene-targeted insects. Our results indicate that the conventional embryonic injection of CRISPR is an effective method to initiate genome editing in T. castaneum. However, it is inefficient by the parental ovary delivery approach.

Effects of embryo injected with ochratoxin A on growth performance, jejunal morphology and barrier of ducklings.

1. This trial investigated the effect on embryo injected with ochratoxin A (OTA) and the growth performance, jejunal morphology and barrier of ducklings to 21 d old.2. Two hundred forty, fertilised eggs were individually weighed and randomly assigned to two groups, a control (CON) and the OTA treatment, according to average egg weight. On d 13 of embryonic development, the treatment group was injected with 8 ng OTA/g egg and the CON group was injected with NaHCO3 solution as a placebo. All newly hatched ducklings were assigned to the CON or OTA group based on the different treatments. Each treatment consisted of six replicates and each included 10 ducklings and the experiment lasted until 21 d of age.3. The results showed that embryos injected with OTA affected the 21 d body weight (BW) and average daily gain (ADG) of ducklings (p < 0.05). OTA exposure increased the relative weights of the liver, pancreas, gizzard, proventriculus and jejunum (p < 0.05); and decreased the relative length of the jejunum of ducklings (p < 0.05). Moreover, jejunal crypt depth increased (p < 0.05) and the villus height-to-crypt depth ratio (Vh/Cd) decreased in the OTA-injected group (p < 0.05). Compared with those in the CON group, the mRNA expression of Zonula Occludens-1; (ZO-1) (p = 0.0582) and Occludin; (p = 0.0687) in the OTA treatment group was downregulated.4. The findings demonstrated that a single low-dose injection of OTA increased body weight and daily gain in ducklings. Moreover, embryo exposure to OTA had negative effects with increased relative weight of organs and the jejunal crypt depth, decreased relative length of the intestine and mRNA expression of tight junctions (ZO-1, Occludin).

Embryo injected with Ochratoxin A induced jejunum injury in ducklings by activating the TLR4 signaling pathway: Involvement of intestinal microbiota.

Ochratoxin A (OTA) is a common mycotoxin that causes intestinal injury in humans and various animal species. OTA may lead to intestinal injury in offspring due to the maternal effect. The aim of this study was to investigate the mechanism of embryo injected with OTA induced jejunum injury in ducklings. The results showed that OTA disrupted the jejunum tight junctions in hatching ducklings, and promoted the secretion of inflammatory cytokines. And this inflammatory response was caused by the activation of the TLR4 signaling pathway. Moreover, embryo injected with OTA could cause damage to the intestinal barrier in 21-day-old ducks, characterized by shortened villi, crypt hyperplasia, disrupted intestinal tight junctions, increased level of LPS in the jejunum, activation of the TLR4 signaling pathway, and increased levels of pro-inflammatory cytokines. Meanwhile, OTA induced oxidative stress in the jejunum. And dysbiosis of gut microbiota was mainly characterized by an increased the relative abundance of Bacteroides, Megamonas, Fournierella, and decreased the relative abundance of Alistipes and Weissella. Interestingly, embryo injected with OTA did not induce these changes in the jejunum of antibiotics-treated 21-day-old ducks. In conclusion, embryo injected with OTA induced jejunum injury in ducklings by activating the TLR4 signaling pathway, which involvement of intestinal microbiota.

Generation of Rabbit Chimeras by Eight-Cell Stage Embryo Injection.

Pluripotent stem cell (PSC) injection to the blastocyst stage embryos is a widely used method to evaluate the pluripotency through chimeric contribution. It is routinely used to produce transgenic mice. However, PSC injection to the blastocyst stage embryos in rabbits is challenging. At this stage, the in vivo developed rabbit blastocysts possess a thick mucin layer that is inhibitory for microinjection, whereas in vitro developed rabbit blastocysts that lack such mucin layer often fail to implant after embryo transfer. In this chapter, we describe a detailed protocol of rabbit chimera production through mucin-free eight-cell stage embryo injection procedure.

Effects of embryo injected with ochratoxins A on hatching quality and jejunum antioxidant capacity of ducks at hatching.

Numerous studies have shown that ochratoxins A (OTA) exerts diverse toxicological effects, namely, hepatotoxicity, nephrotoxicity, genotoxicity, enterotoxicity, and immunotoxicity. The main objective of this study was to investigate the influence of embryonic exposure to OTA by different injection times and OTA doses on hatching quality and jejunal antioxidant capacity of ducks at hatching. In total, 480 fertilized eggs were weighed and randomly assigned into a 4 × 4 factorial design including four OTA doses (0, 2, 4, and 8 ng/g egg) on 8, 13, 18, and 23 of embryonic development (E8, E13, E18, and E23). Each treatment included 6 repeats with 5 eggs per repeat. The results showed that the injection time affected the hatching weight (P < 0.0001). The relative length of the jejunum and ileum on E18 and E23 was lower than on E8 and E13 (P < 0.05). Injection time, doses, and their interaction had no effect on jejunum morphology, namely, villous height (Vh), crypt depth (Cd), and villous height/crypt depth ratio Vh/Cd (P > 0.05). The injection time affected the activities of Superoxide dismutase (SOD) (P < 0.0001), total antioxidant capacity (T-AOC) (P < 0.05) and the malondialdehyde (MDA) content (P < 0.0001). The activity of SOD and T-AOC activities in the jejunum of ducklings injected with OTA at the E8 and E13 was lower than that injected at the E18 (P < 0.05). The highest MDA content was observed in ducklings injected with OTA at the E13 (P < 0.05). The injection time (P < 0.0001), OTA doses and their interaction affected the contents of IL-1ß (P < 0.05), which significantly increased especially on E13. In conclusion, the embryo injected with ochratoxins A affected the hatching weight, the relative length of jejunum and ileum, decreased the antioxidant capacity and increased the content of proinflammatory cytokine IL-1ß of the jejunum.

Functional Analysis of Individual piRNAs in Aedes aegypti Cells and Embryos Using Antisense Oligonucleotides.

In insects, PIWI-interacting (pi)RNAs fulfill versatile regulatory functions inside and outside the germline, including posttranscriptional repression of transposable elements and regulation of gene expression. Canonically, piRNAs act-and have been studied-as a conglomerate of several thousand sequences that cooperatively silence target RNAs. Interestingly, however, an increasing number of studies have demonstrated that individual piRNAs can have profound biological activity as a unique piRNA sequence. Prime examples are the tapiR1 and 2 piRNAs, which mediate target RNA degradation in the developing embryo of Aedes mosquitoes. To study such outstanding individual piRNA species, we describe here a method to interfere with RNA target silencing using antisense oligonucleotides in cell culture as well as in mosquito pre-blastoderm embryos. Although the method has been established for Aedes mosquitoes, it can likely be adapted for use in other invertebrate species as well.

One-step base editing in multiple genes by direct embryo injection for pig trait improvement.

The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors (CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations. Our process resulted in a stop codon in CD163 and myostatin (MSTN) and introduced a beneficial allele in insulin-like growth factor-2 (IGF2). The pigs showed disrupted expression of CD163 and MSTN and increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection, providing a potential new route to produce elite pig breeders.

Genetic Manipulation of Ticks: A Paradigm Shift in Tick and Tick-Borne Diseases Research.

Ticks are obligate hematophagous arthropods that are distributed worldwide and are one of the most important vectors of pathogens affecting humans and animals. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind other arthropod vectors, such as mosquitoes. This is largely because of challenges in applying functional genomics and genetic tools to the idiosyncrasies unique to tick biology, particularly techniques for stable genetic transformations. CRISPR-Cas9 is transforming non-model organism research; however, successful germline editing has yet to be accomplished in ticks. Here, we review the ancillary methods needed for transgenic tick development and the use of CRISPR/Cas9, the most promising gene-editing approach, for tick genetic transformation.

Pluripotent stem cells secrete Activin A to improve their epiblast competency after injection into recipient embryos.

It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a "dose effect". Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development.