Clustered Regularly Interspaced Short Palindromic repeats/CRISPR-associated 9 Research Articles

PTTH-Torso Signaling System Controls Developmental Timing, Body Size, and Reproduction through Regulating Ecdysone Homeostasis in the Brown Planthopper, Nilaparvata lugens.

In holometabolous insects, such as Drosophila and Bombyx, prothoracicotropic hormone (PTTH) is well established to be critical in controlling developmental transitions and metamorphosis by stimulating the biosynthesis of ecdysone in the prothoracic glands (PGs). However, the physiological role of PTTH and the receptor Torso in hemimetabolous insects remains largely unexplored. In this study, homozygous PTTH- and Torso-null mutants of the brown planthopper (BPH), Nilaparvata lugens, were successfully generated by employing clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR-Cas9). Further characterization showed that both NlPTTH-/- and NlTorso-/- mutants exhibited prolonged nymphal duration and increased final adult size. Enzyme-linked immunosorbent assay (ELISA) revealed that NlPTTH-/- and NlTorso-/- mutants exhibited a significant reduction in 20-hydroxyecdysone (20E) in fifth-instar nymphs at 48 h post-ecdysis compared to Wt controls. Furthermore, our results indicated that both NlPTTH-/- and NlTorso-/- mutants had shortened lifespan, reduced female fecundity, and reduced egg hatching rates in adults. These findings suggest a conserved role for the PTTH-Torso signaling system in the regulation of developmental transitions by stimulating ecdysone biosynthesis in hemimetabolous insects.

One-step in vivo gene knock-out in porcine embryos using recombinant adeno-associated viruses.

Introduction: Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) technology is a widely employed tool for generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due to its reliance on intricate micromanipulation equipment and specialized techniques, resulting in high costs and time-consuming procedures. This study aims to introduce a novel one-step approach for generating genetically modified pigs by transducing CRISPR/Cas9 components into pre-implantation porcine embryos through oviductal injection of recombinant adeno-associated viruses (rAAV). Methods: We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP to screen for rAAV serotypes that efficiently target porcine embryos, and then, to achieve efficient expression of CRISPR/Cas9 in vivo for a short period, we packaged sgRNAs targeting the GHR genes to self-complementary adeno-associated virus (scAAV), and Cas9 proteins to single-stranded adeno-associated virus (ssAAV). The efficiency of porcine embryos -based editing was then validated in vitro. The feasibility of this one-step method to produce gene-edited pigs using rAAV-CRISPR/Cas9 oviductal injection into sows within 24h of conception was then validated. Results: Our research firstly establishes the efficient delivery of CRISPR/Cas9 to pig zygotes, both in vivo and in vitro, using rAAV6. Successful gene editing in pigs was achieved through oviductal injection of rAAV-CRISPR/Cas9. Conclusion: This method circumvents the intricate procedures involved in in vitro embryo manipulation and embryo transfers, providing a straightforward and cost-effective approach for the production of gene-edited pigs.

Exploring the potential of CRISPR/Cas genome editing for vegetable crop improvement: An overview of challenges and approaches.

Vegetables provide many nutrients in the form of fiber, vitamins, and minerals, which make them an important part of our diet. Numerous biotic and abiotic stresses can affect crop growth, quality, and yield. Traditional and modern breeding strategies to improve plant traits are slow and resource intensive. Therefore, it is necessary to find new approaches for crop improvement. Clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (CRISPR/Cas9) is a genome editing tool that can be used to modify targeted genes for desirable traits with greater efficiency and accuracy. By using CRISPR/Cas9 editing to precisely mutate key genes, it is possible to rapidly generate new germplasm resources for the promotion of important agronomic traits. This is made possible by the availability of whole genome sequencing data and information on the function of genes responsible for important traits. In addition, CRISPR/Cas9 systems have revolutionized agriculture, making genome editing more versatile. Currently, genome editing of vegetable crops is limited to a few vegetable varieties (tomato, sweet potato, potato, carrot, squash, eggplant, etc.) due to lack of regeneration protocols and sufficient genome sequencing data. In this article, we summarize recent studies on the application of CRISPR/Cas9 in improving vegetable trait development and the potential for future improvement.

Advances in Plant Epigenome Editing Research and Its Application in Plants

Plant epistatic regulation is the DNA methylation, non-coding RNA regulation, and histone modification of gene sequences without altering the genome sequence, thus regulating gene expression patterns and the growth process of plants to produce heritable changes. Epistatic regulation in plants can regulate plant responses to different environmental stresses, regulate fruit growth and development, etc. Genome editing can effectively improve plant genetic efficiency by targeting the design and efficient editing of genome-specific loci with specific nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9). As research progresses, the CRISPR/Cas9 system has been widely used in crop breeding, gene expression, and epistatic modification due to its high editing efficiency and rapid translation of results. In this review, we summarize the recent progress of CRISPR/Cas9 in epigenome editing and look forward to the future development direction of this system in plant epigenetic modification to provide a reference for the application of CRISPR/Cas9 in genome editing.

Evolution of the WRKY66 Gene Family and Its Mutations Generated by the CRISPR/Cas9 System Increase the Sensitivity to Salt Stress in Arabidopsis.

Group Ⅲ WRKY transcription factors (TFs) play pivotal roles in responding to the diverse abiotic stress and secondary metabolism of plants. However, the evolution and function of WRKY66 remains unclear. Here, WRKY66 homologs were traced back to the origin of terrestrial plants and found to have been subjected to both motifs' gain and loss, and purifying selection. A phylogenetic analysis showed that 145 WRKY66 genes could be divided into three main clades (Clade A-C). The substitution rate tests indicated that the WRKY66 lineage was significantly different from others. A sequence analysis displayed that the WRKY66 homologs had conserved WRKY and C2HC motifs with higher proportions of crucial amino acid residues in the average abundance. The AtWRKY66 is a nuclear protein, salt- and ABA- inducible transcription activator. Simultaneously, under salt stress and ABA treatments, the superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, as well as the seed germination rates of Atwrky66-knockdown plants generated by the clustered, regularly interspaced, short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system, were all lower than those of wild type (WT) plants, but the relative electrolyte leakage (REL) was higher, indicating the increased sensitivities of the knockdown plants to the salt stress and ABA treatments. Moreover, RNA-seq and qRT-PCR analyses revealed that several regulatory genes in the ABA-mediated signaling pathway involved in stress response of the knockdown plants were significantly regulated, being evidenced by the more moderate expressions of the genes. Therefore, the AtWRKY66 likely acts as a positive regulator in the salt stress response, which may be involved in an ABA-mediated signaling pathway.

FoxO is required for optimal fitness of the migratory brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

The forkhead box O (FoxO) protein is the main transcriptional effector downstream of the insulin/insulin-like signaling pathway and regulates many developmental and physiological processes. Holometabolous insects with loss-of-function mutations in FoxO exhibit phenotypes distinct from those of hemimetabolous insects in which RNA interference was used. Despite the functional importance of FoxO, whether hemimetabolous insects share an evolutionally conserved function of FoxO with holometabolous insects remains to be clarified. We used the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) genome editing-system to establish a homozygous FoxO-null mutant (NlFoxO4E ) of the wing-dimorphic brown planthopper (BPH) Nilaparvata lugens, an economically important insect pest of rice fields. The phenotypes of NlFoxO4E mutants included extended nymphal duration, shortened lifespan, reduced reproduction, and decreased stress resistance. In addition, depletion of NlFoxO promoted cell proliferation in wing buds and led to 100% long-winged morphs, in stark contrast to short-winged wild-type BPHs. These findings indicate that NlFoxO is highly functionally conserved with its counterpart in holometabolous insects, and is required for optimal fitness of N. lugens. The insights from FoxO studies may facilitate the identification of potential target genes for BPH control applications.

CRISPR/Cas9 technology and its application in horticultural crops

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) system has recently become one popular technology due to its efficiency, precision, and simplicity compared with other genome editing tools such as Zinc Finger Nucleases (ZFNs) and Transcription Activator Like Effector Nucleases (TALENs). Horticultural crops provide energy and health-keeping nutrients to humankind. Genome-editing technology has become widely adopted in horticultural breeding with the increasing demand for high yield and better-quality horticultural crops. Here, we describe the CRISPR/Cas9 system construction, its optimization, including sgRNA promoter, sgRNA design, Cas9 protein promoter, SpCas9 variants and orthologs, and vector delivery methods. We also summarized the application of this technology in horticultural plants for stress responses enhancement, fruit quality improvement, and cultivation traits modification. This detailed review was compiled to help establish comprehensive understanding of the CRISPR/Cas9 systems and provide a reference for further developing this technology to manipulate horticultural plant traits effectively.

Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out.

Among the three existing targeted gene editing technologies, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9), the latter is widely used owing to its simplicity, efficiency, and low cost. Here, we routinely infected A172 and U251 cells with lentiviral vectors, in which aquaporin-8 (AQP8) was knocked out using CRISPR/Cas9. Our results indicated that cryopreservation did not significantly alter the viral infection efficiency, but influenced AQP8 expression in the infected cells at both protein and mRNA levels compared with the non-cryopreserved samples. Further, AQP8 expression at protein and mRNA levels in recovered cryopreserved infected cells did not significantly differ from those in the blank and negative controls, indicating that the lentivirus was still infectious at low temperatures. However, it failed to release the AQP8-targeting guide RNA in the infected cells, or the guide RNA was released, but underwent changes that caused it to malfunction in the cells with CRISPR/Cas9-mediated AQP8 knock-out. Our findings possibly provide some insights into the reliability of lentiviruses as CRISPR/Cas9 vectors.

Macrophages regulates the transition of pericyte to peritoneal fibrosis through the GSDMD/IL-1β axis

BackgroundEnd stage renal disease (ESRD) has caused public health problem with high prevalence worldwide. Peritoneum from peritoneal dialysis patients with ESRD can induce pathological changes of the peritoneum, including fibrosis. The trans-differentiation of pericytes has been found to be closely associated with inflammatory diseases, such as organ fibrosis. However, the function of macrophages in regulating the transition of pericyte to peritoneal fibrosis is unclear. MethodsHistological examination was conducted using Hematoxylin and eosin (HE) staining and Masson’s trichrome staining. The protein levels were determined via western blot. Enzyme-linked immunosorbent assay (ELISA) was used to examine IL-1β concentrations. Gasdermin D (GSDMD) was knocked out in mice by Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9). ResultsMice receiving dextrose peritoneal dialysate displayed mesothelial cell monolayer loss and thickness of submesothelial compact zone increase. Moreover, dextrose peritoneal dialysate treatment up-regulated GSDMD expression. GSDMD knockdown inhibited IL-1β production in macrophages. Further, pericytes were treated with cultural supernatant from macrophages. We found that GSDMD knockdown suppressed fibrosis and vascular endothelial growth factor (VEGF)/phosphoinositide 3-kinase (PI3K) pathway in pericytes. In addition, GSDMD were knocked out in mice using CRISPR/Cas9. The histological examinations revealed that GSDMD−/− alleviated the damage of peritoneal tissue and thickness of submesothelial compact zone. GSDMD−/− attenuated interleukin-1beta (IL-1β) level and peritoneal fibrosis induced by dextrose peritoneal dialysate treatment in pericytes in vivo. ConclusionThese results demonstrated that macrophages can regulate the transition of pericyte to peritoneal fibrosis via the GSDMD/IL-1β axis, which provides a new therapeutic target.

No apparent p53 activation in CRISPR-engineered gene-edited rabbits.

Clustered regularly interspaced short palindromic repeats‐CRISPR‐associated 9 (CRISPR‐Cas9) and base editors (BEs) are revolutionary gene‐editing technology that has been widely utilized in biology, biotechnology and medicine. However, recent reports show that CRISPR‐Cas9‐mediated genome editing can induce a p53‐mediated stress response and cell cycle arrest in human cells, while not illustrated in gene‐editing animals. In the study, to verify whether there is a phenomenon of p53 activation, by analysing nine gene‐edited rabbits using CRISPR‐Cas9 and BEs, we provide the first evidence that no apparent p53 expression changes in those rabbits generated by Cas9 or BE‐edited, suggesting that p53 may not need to consider for application in gene‐edited animals.

Relationship between DNA mismatch repair and CRISPR/Cas9-mediated knock-in in the bovine β-casein gene locus.

ObjectiveEfficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2).MethodsBovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl2 for 24 hours. After 3 days of CdCl2 treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR).ResultsTreatment with CdCl2 decreased the mRNA expression of RAD51 and MMR-related genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency.ConclusionTreatment with CdCl2 inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

TXNIP positively regulates the autophagy and apoptosis in the rat müller cell of diabetic retinopathy

AimsDiabetic retinopathy (DR) can cause vision loss in patients with diabetes. The present study evaluated the expression of thioredoxin interacting protein (TXNIP) and investigated the role of TXNIP in autophagy and apoptosis of DR. Main methodsReverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to measure the expression level of the targets. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/cas9) method was applied for knockout of TXNIP. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay and flow cytometry were utilized to detect the apoptosis. Cell Counting Kit-8 (CCK-8) assay was used to evaluate the cell viability. EdU assay was carried out to measure the cell proliferation ability. Retinal immunohistochemistry, retinal frozen section immunofluorescence as well as the electroretinogram (ERG) recording were implemented to detect the function of the retina. Key findingsTXNIP was up-regulated under hyperglycemic condition both in vivo and in vitro. Overexpression of TXNIP activated the autophagy and apoptosis in the rat müller cell. Knockout of TXNIP reduced the autophagy and apoptosis in the rat müller cell under high glucose condition. TXNIP positively regulates autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway. Knockdown of TXNIP improved the visual response to light stimulus of DR. SignificanceOur study unraveled for the first time that TXNIP positively regulates the autophagy in rat müller cell under high glucose condition by inhibiting the PI3K/AKT/mTOR signaling pathway, providing a novel understanding in the pathogenesis of DR and suggesting a potential new therapeutic target of DR.

A novel strategy for creating a new system of third-generation hybrid rice technology using a cytoplasmic sterility gene and a genic male-sterile gene.

SummaryHeterosis utilization is the most effective way to improve rice yields. The cytoplasmic male‐sterility (CMS) and photoperiod/thermosensitive genic male‐sterility (PTGMS) systems have been widely used in rice production. However, the rate of resource utilization for the CMS system hybrid rice is low, and the hybrid seed production for the PTGMS system is affected by the environment. The technical limitations of these two breeding methods restrict the rapid development of hybrid rice. The advantages of the genic male‐sterility (GMS) rice, such as stable sterility and free combination, can fill the gaps of the first two generations of hybrid rice technology. At present, the third‐generation hybrid rice breeding technology is being used to realize the application of GMS materials in hybrid rice. This study aimed to use an artificial CMS gene as a pollen killer to create a smart sterile line for hybrid rice production. The clustered regularly interspaced short palindromic repeats/CRISPR‐associated 9 (CRISPR/Cas9) technology was used to successfully obtain a CYP703A3‐deficient male‐sterile mutant containing no genetically modified component in the genetic background of indica 9311. Through young ear callus transformation, this mutant was transformed with three sets of element‐linked expression vectors, including pollen fertility restoration gene CYP703A3, pollen‐lethality gene orfH79 and selection marker gene DsRed2. The maintainer 9311‐3B with stable inheritance was obtained, which could realize the batch breeding of GMS materials. Further, the sterile line 9311‐3A and restorer lines were used for hybridization, and a batch of superior combinations of hybrid rice was obtained.

CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera)

Grapevine (Vitis vinifera), one of the most economically important fruit crops in the world, suffers significant yield losses from powdery mildew, a major fungal disease caused by Erysiphe necator. In addition to suppressing host immunity, phytopathogens modulate host proteins termed susceptibility (S) factors to promote their proliferation in plants. In this study, CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable the targeted mutagenesis of MLO (mildew resistance Locus O) family genes that are thought to serve as S factors for powdery mildew fungi. Small deletions or insertions were induced in one or both alleles of two grapevine MLO genes, VvMLO3 and VvMLO4, in the transgenic plantlets of the powdery mildew-susceptible cultivar Thompson Seedless. The editing efficiency achieved with different CRISPR/Cas9 constructs varied from 0 to 38.5%. Among the 20 VvMLO3/4-edited lines obtained, one was homozygous for a single mutation, three harbored biallelic mutations, seven were heterozygous for the mutations, and nine were chimeric, as indicated by the presence of more than two mutated alleles in each line. Six of the 20 VvMLO3/4-edited grapevine lines showed normal growth, while the remaining lines exhibited senescence-like chlorosis and necrosis. Importantly, four VvMLO3-edited lines showed enhanced resistance to powdery mildew, which was associated with host cell death, cell wall apposition (CWA) and H2O2 accumulation. Taken together, our results demonstrate that CRISPR/Cas9 genome-editing technology can be successfully used to induce targeted mutations in genes of interest to improve traits of economic importance, such as disease resistance in grapevines.

Optimization of the protoplast transient expression system for gene functional studies in strawberry (Fragaria vesca)

Polyethylene glycol (PEG)-mediated transient expression system in plant protoplasts has been widely used in a variety of plants for gene function characterization. However, such a system has not been developed for strawberry. In this study, we report a method for obtaining high quality and high yield protoplasts from strawberry leaves. In addition, we developed an efficient transient gene expression system based on the PEG-mediated method, and the system has been successfully applied to studies of protein expression, protein subcellular localization and protein–protein interaction in strawberry. Furthermore, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable targeted mutagenesis of the FvTCP17 gene in strawberry protoplasts. Taken together, our results demonstrate the potential of this highly efficient mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9-mediated genome editing in strawberry. We report a highly efficient mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9‐mediated genome editing in strawberry.

Effective restoration of dystrophin expression in iPSC Mdx-derived muscle progenitor cells using the CRISPR/Cas9 system and homology-directed repair technology

Duchenne muscular dystrophy (DMD) is a progressive myopathic disease caused by mutations in the gene encoding dystrophin protein that eventually leads to the exhaustion of myogenic progenitor cells (MPC). Autologous induced pluripotent stem cells (iPSCs) provide an endless source of MPC, which can potentially replenish the progenitor cell pool, repair muscle damage, and prevent DMD progression. Deletion of mutant exon 23 (ΔEx23) with clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology can correct dystrophin gene expression in iPSCs. However, successful exon23 deletion and clonal isolation are very inefficient (~3%), and manual selection of each iPSC clone and genotyping to identify ΔEx23 is labor-intensive. To overcome these obstacles, we added a homology-directed repair (HDR) donor vector, which carries floxed fluorescent protein and antibiotic selection genes, thus allowing us to identify ΔEx23 iPSC with donor selective gene integration. Our results indicate that the HDR-mediated targeted integration enables ΔEx23 iPSC identification; the HDR donor vector increased the recognition efficiency of clonal isolation (>90% as confirmed by Sanger sequencing). After removal of the inserted genes by Cre-mediated recombination followed by doxycycline (Dox)-induced MyoD induction, ΔEx23 iPSC differentiated into MPC with restored dystrophin expression in vitro. Importantly, transplanted ΔEx23 iPSC-MPC express dystrophin in the muscles of a mouse model of DMD (Mdx mice). In conclusion, the use of HDR donor vector increased the efficiency of ΔEx23 gene correction by CRISPR/Cas9, and facilitate the identification of successfully edited iPSC clones for cell therapy of DMD.

Generation and characterisation of a COV434 cell clone carrying a monoallelic FecBB mutation introduced by CRISPR/Cas9

The FecBB mutation, which increases the ovulation quota, was identified in a hyperprolific sheep breed, and is associated with a single point mutation (c.A746G; p.Q249R) in bone morphogenetic protein receptor-type 1B (BMPR1B). However, the mechanism of action of the FecBB mutation remains unclear. Here, we describe the application of clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) to introduce the FecBB mutation into the human granulosa cell line COV434 via homology-directed repair (HDR), using single-stranded oligodeoxynucleotides (ssODNs) as template. Upon screening single cell-derived clones, we found one clone containing the FecBB mutation on one allele and knockout of BMPR1B on the other allele, and another clone harbouring an in-frame deletion on one allele and knockout of BMPR1B on the other. These two clones were subjected to functional analysis. We found that the FecBB mutation and knockout of BMPR1B could upregulate the expression of cumulus expansion-related genes, potentially mediated by enhanced activation of the Sma- and Mad-related 1/5 (SMAD1/5) pathway. The FecBB mutation, an in-frame mutation, and knockout of BMPR1B could impair the synthesis of oestradiol in COV434 cells, possibly through inhibition of the SMAD2/3 pathway. Our study provides a useful cell model for further investigation into the mechanism of action of the FecBB mutation.

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmdmdx mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.

Efficient somatic and germline genome engineering of Bactrocera dorsalis by the CRISPR/Cas9 system.

Bactrocera dorsalis (Hendel), a very destructive insect pest of many fruits and vegetables, is widespread in many Asian countries. To facilitate control of this pest, it is essential to investigate its genetics and gene function using targeted gene disruption. Here, we describe successful targeted mutagenesis of the white and transformer genes in B. dorsalis through use of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. Co-injection of the white sgRNA and Cas9 mRNA into B. dorsalis embryos caused eye color change, and the white mutations in the germline were heritable. CRISPR-mediated knockout of the sex determination gene transformer (tra) in B. dorsalis resulted in a male-biased sex ratio and adult flies with abnormal outer and interior reproductive organs. Small indels and substitutions were induced by CRIRPR for both genes. Our data demonstrate that somatic and germline genome engineering of the pest B. dorsalis can be performed efficiently using the CRISPR/Cas9 system, opening the door to the use of the CRISPR-mediated method for functional annotations of genes in B. dorsalis and for its population control using, for example, such as gene drive. © 2018 Society of Chemical Industry.