Electroporation-based CRISPR/Cas9 Gene Editing in Haliotis Discus Hannai.

Creating transgenic mosquitoes allows for mechanistic studies of basic mosquito biology and the development of novel vector control strategies. CRISPR-Cas9 gene editing has revolutionized gene editing, including in mosquitoes. This protocol details part of the gene editing process of Aedes aegypti mosquitoes via CRISPR-Cas9, through testing and validating single-guide RNAs (sgRNAs). Gene editing activity varies depending on the sequence of sgRNAs used, so validation of sgRNA activity should be done before large-scale generation of mutants or transgenics. sgRNA is designed using online tools and synthesized in <1 h. Once mutants or transgenics are generated via embryo microinjection, sgRNA activity is validated by quick genotyping polymerase chain reaction (PCR) and DNA sequencing.

The farming of livestock has a critical role to play in global nutritional security and poverty alleviation. To meet these goals through more efficient, environmentally sustainable and animal welfare focused means, gene editing technologies could be integrated into current breeding programs. A common issue with gene editing in livestock zygotes is the high incidence of genetic mosaicism. Genetic mosaicism, characterised by a single individual carrying distinct genotypes in different cell lineages, can lead to inconsistent presentation of a desired trait phenotypically, or the absence of the intended genotype in the animal’s germline. This review explores the present status of genetic mosaicism associated with CRISPR-Cas9 gene editing in cattle, sheep, and pigs, and identifies four areas for refinement; (1) the type of CRISPR-Cas9 genome editor used; (2) the CRISPR-Cas9 formats and timing of gene editing during embryonic development; (3) the method used to deliver the genome editor and (4) the genetic screening strategies applied. We also discuss alternatives to direct zygote gene editing, including surrogate sire technology and blastomere separation, which circumvent the production of mosaic offspring. By exploring these avenues for reducing mosaicism, gene editing protocols in livestock could become more efficient and effective, which will ultimately pave the way for traits to be introduced that improve animal welfare standards and help address gaps in the security of global nutrition access.

Background: Gene editing (GE) can be considered among the most promising strategies to correct hereditary mutations in a variety of monogenetic diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9 gene editing can be proposed for efficient correction of the β039-thalassemia mutation. GE is suitable for personalized approaches in precision medicine for β-thalassemia [1]. Aims: On the other hand, robust evidence demonstrate that increased production of fetal hemoglobin (HbF) can be beneficial for β-thalassemia patients. This can be obtained also using CRISPR-Cas9 based protocols. In this case, the objective of the gene editing strategy is (a) the silencing of transcriptional repressors of the γ-globin genes (such as BCL11A), (b) the disruption of their regulatory binding sites within the γ-globin genes, or (c) the induction of natural hereditary persistence of fetal hemoglobin mutations in the γ-globin gene or the b-like-globin gene cluster. Our study aimed to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols based on the use of repositioned drugs. Results: Gene Editing of the β039-thalassemia mutation was obtained by the CRISPR-Cas9 method; gene correction and transcription of the corrected gene have been analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; production of HbA and HbF was studied by HPLC and Western blotting. For HbF induction, the repurposed drug sirolimus (rapamycin) was used. The data obtained conclusively demonstrate that maximal production of HbA and HbF is obtained in GE-corrected, sirolimus-induced erythroid progenitors from β039-thalassemia patients. In conclusion, GE and HbF in-duction might be used in combination in order to achieve the de novo production of HbA together with increased production of induced HbF. Acknowledgements This study was sustained by the Wellcome Trust (innovator award 208872/Z/17/Z) and by AIFA (AIFA-2016-02364887). The research leading to these results has received funding also from the UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of Βeta-Thalassemia; no. 306201-FP7-HEALTH-2012-INNOVATION-1). We thanks A.L.T. (Associazione per la lotta alla Talassemia) “Rino Vullo” - Ferrara, and A.V.L.T. (Associazione Veneta per la Lotta alla Talassemia) “Elio Zago” - APS – Rovigo and Association “Tutti per Chiara”, Montagnana, PD, Italy. Reference 1. Cosenza L.C., Gasparello J., Romanini N., Zurlo M., Zuccato C., Gambari R., Finotti A. Efficient CRISPR-Cas9-based genome editing of β-globin gene on erythroid cells from homozygous β039-thalassemia patients. Mol Ther Methods Clin Dev 2021;21:507−523. https://doi.org/10.1016/j.omtm.2021.03.025

A humanized knockin mouse model of Duchenne muscular dystrophy and its correction by CRISPR-Cas9 therapeutic gene editing

Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the β039-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with β-thalassemia. The aim of our study was to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols. The gene editing of the β039-globin mutation was obtained using a CRISPR-Cas9-based experimental strategy; the correction of the gene sequence and the transcription of the corrected gene were analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; the relative content of HbA and HbF was studied by high-performance liquid chromatography (HPLC) and Western blotting. For HbF induction, the repurposed drug rapamycin was used. The data obtained conclusively demonstrate that the maximal production of HbA and HbF is obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from β039-thalassemia patients. In conclusion, GE and HbF induction might be used in combination in order to achieve the de novo production of HbA together with an increase in induced HbF.

Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.

T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first gene-modified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

The settlement, early growth and survival of the larval abalone Haliotis discus hannai in response to eight monospecific benthic diatoms were examined in the laboratory. Postlarvae showed active settling and feeding behaviour in all diatom species and in naturally occurring diatoms. Larval settlement rates differed significantly between experimental substrata after 24 h and 48 h. Nitzschia sp. (96.67 %), Hantzschia amphioxys var. leptocephala (95.00 %) and Navicula seminulum (90.00 %) strongly induced larval settlement of H. discus hannai. Postlarvae could feed on benthic diatoms (< 36 μm in shell length) with both weak and strong adhesion on the 4th day after settlement. Greatest growth (shell length) occurred on Nitzschia sp. (786.84 μm ± 2.50 SE and 773.09 μm ± 2.09 SE). Survival of postlarvae was also greatest on Nitzschia sp. (95.33 % ± 1.45 SE). These results indicate the effectiveness of Nitzschia sp., H. amphioxys var. leptocephala, N. seminulum, Rhaphoneis surirella and Navicula corymbosa as single species over natural diatoms in larval settlement and postlarvae growth of H. discus hannai. Thus, Nitzschia sp. has the best potential diet for larval settlement and postlarvae growth of H. discus hannai. H. amphioxys var. leptocephala and N. seminulum can be used as cues to induce larvae settlement, and R. surirella and N. corymbosa can be used as food for growing postlarval.

Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L. and Haliotis discus hannai Ino. V. The role of polyunsaturated fatty acids of macroalgae in abalone nutrition

Performance of triploid Haliotis discus hannai cultured in a subtropical area using sea-based suspended systems

Hemolymph and transcriptome analysis to understand innate immune responses to hypoxia in Pacific abalone

Molecular mechanisms underlying the vulnerability of Pacific abalone (Haliotis discus hannai) to Vibrio harveyi infection at higher water temperature.

Vibrio harveyi is capable of inducing vibriosis in Haliotis discus hannai and causes enormous economic losses to marine mollusks. The intestinal microbiota plays critical roles in immune homeostasis and provides essential health benefits or poses risks to the host. However, there is still little information about the intestinal microbiota responses of H. discus hannai to V. harveyi infection. We investigated the variations in the mortality rate, pathological changes, and intestinal microbiota communities of abalone after exposure to V. harveyi . The present study investigated the intestinal microbial response to acute infection of H. discus hannai with V. harveyi at 12 hours post-infection (hpi), 32 hpi and 60 hpi by 16S rRNA gene sequencing. The results indicated that prolongation of infection exerts a significant impact on the intestinal microbial community, and within-group variability gradually decreased. At 32 hpi and 60 hpi, the relative abundance of Mycoplasmataceae and Ruminococcaceae was significantly decreased, while Vibrionaceae and Fusobacteriaceae exhibited a significant increase in relative abundance. The results demonstrated that infection of abalone with V. harveyi promoted the proliferation of pathogenic bacteria in the intestinal microbiota and thereby facilitated the development of disease in the abalones. This study aimed to explore the role of the intestinal microbiota in the pathogenic mechanism of V. harveyi infection, and provide new scientific insights into disease courses of abalones.

Pacific abalone, Haliotis discus hannai, is an economically important marine mollusk species and an important model animal for studies on ecological, fertilization and developmental biology. While embryonic injection and genome editing have been wildly used in gene function study and trait improvement in many species, they have not been developed in abalones. In this study, we reported an effective method to inject exogenous materials in H. discus hannai unfertilized eggs. The injected eggs could be fertilized at a ratio of 52.6% ± 5.9% and hatch at a ratio of 14.6% ± 1.6%. On the base of this, we further developed an efficient genome editing approach in this species with the transcription activator-like effector nuclease (TALEN) technique. Two TALEN pairs targeting the coding sequence of the abalone nodal gene were assembled and tested. While one of the TALEN pairs showed no detectable mutation efficacy, the other one generated mutations in 50% of the targeted loci. The mutation includes small insertions and deletions and base pair replacements like that reported in other species when the TALEN method was applied. Overall, this is the first study to demonstrate site-specific genome editing in abalone. This work can serve as a reference for future studies focusing on the functional genomics in mollusks.

A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells