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Abstract Background Enhancer reprogramming plays a significant role in the heterogeneity of cancer. However, we have limited knowledge about the impact of chromatin remodeling in B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) patients, and how it affects tumorigenesis and drug response. Our research focuses on investigating the role of enhancers in sustaining oncogenic transformation in children with BCP-ALL. Methods We used ATAC-seq to study the accessibility of chromatin in pediatric BCP-ALL at three different stages—onset, remission, and relapse. Using a combination of computational and experimental methods, we were able to analyze the accessibility landscape and focus on the most significant cis-regulatory sites. These sites were then functionally validated through the use of Promoter capture Hi-C in a primary cell line model called LAL-B, followed by RNA-seq and genomic deletion of target sites using CRISPR-Cas9 editing. Results We found that enhancer activity changes during cancer progression and is mediated by the production of enhancer RNAs (eRNAs). CRISPR-Cas9-mediated validation of previously unknown eRNA productive enhancers demonstrated their capability to control the oncogenic activities of the MYB and DCTD genes. Conclusions Our findings directly support the notion that productive enhancer engagement is a crucial determinant of the BCP-ALL and highlight the potential of enhancers as therapeutic targets in pediatric BCP-ALL.

The role of messenger RNA (mRNA) in biological systems is extremely versatile. However, it’s extremely short half-life poses a fundamental restriction on its application. Moreover, the translation efficiency of mRNA is also limited. On the contrary, circular RNAs, also known as circRNAs, are a common and stable form of RNA found in eukaryotic cells. These molecules are synthesized via back-splicing. Both synthetic circRNAs and certain endogenous circRNAs have the potential to encode proteins, hence suggesting the potential of circRNA as a gene expression machinery. Herein, we aim to summarize all engineering aspects that allow exogenous circular RNA (circRNA) to prolong the time that proteins are expressed from full-length RNA signals. This review presents a systematic engineering approach that have been devised to efficiently assemble circRNAs and evaluate several aspects that have an impact on protein production derived from. We have also reviewed how optimization of the key components of circRNAs, including the topology of vector, 5′ and 3′ untranslated sections, entrance site of the internal ribosome, and engineered aptamers could be efficiently impacting the translation machinery for molecular and metabolic reprogramming. Collectively, molecular and metabolic reprogramming present a novel way of regulating distinctive cellular features, for instance growth traits to neoplastic cells, and offer new possibilities for therapeutic inventions. [ABSTRACT FROM AUTHOR]

Background: Enhancer reprogramming plays a significant role in the heterogeneity of cancer. However, we have limited knowledge about the impact of chromatin remodeling in B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) patients, and how it affects tumorigenesis and drug response. Our research focuses on investigating the role of enhancers in sustaining oncogenic transformation in children with BCP-ALL. Methods: We used ATAC-seq to study the accessibility of chromatin in pediatric BCP-ALL at three different stages—onset, remission, and relapse. Using a combination of computational and experimental methods, we were able to analyze the accessibility landscape and focus on the most significant cis-regulatory sites. These sites were then functionally validated through the use of Promoter capture Hi-C in a primary cell line model called LAL-B, followed by RNA-seq and genomic deletion of target sites using CRISPR-Cas9 editing. Results: We found that enhancer activity changes during cancer progression and is mediated by the production of enhancer RNAs (eRNAs). CRISPR-Cas9-mediated validation of previously unknown eRNA productive enhancers demonstrated their capability to control the oncogenic activities of the MYB and DCTD genes. Conclusions: Our findings directly support the notion that productive enhancer engagement is a crucial determinant of the BCP-ALL and highlight the potential of enhancers as therapeutic targets in pediatric BCP-ALL. [ABSTRACT FROM AUTHOR]

Abstract Background CRISPR/Cas9 system to treat human-related diseases has achieved significant results and, even if its potential application in cancer research is improving, the application of this approach in clinical practice is still a nascent technology. Main body CRISPR/Cas9 technology is not yet used as a single therapy to treat tumors but it can be combined with traditional treatment strategies to provide personalized gene therapy for patients. The combination with chemotherapy, radiation and immunotherapy has been proven to be a powerful means of screening, identifying, validating and correcting tumor targets. Recently, CRISPR/Cas9 technology and CAR T-cell therapies have been integrated to open novel opportunities for the production of more efficient CAR T-cells for all patients. GMP-compatible equipment and reagents are already available for several clinical-grade systems at present, creating the basis and framework for the accelerated development of novel treatment methods. Conclusion Here we will provide a comprehensive collection of the actual GMP-grade CRISPR/Cas9-mediated approaches used to support cancer therapy highlighting how this technology is opening new opportunities for treating tumors.

Background: CRISPR/Cas9 system to treat human-related diseases has achieved significant results and, even if its potential application in cancer research is improving, the application of this approach in clinical practice is still a nascent technology. Main body: CRISPR/Cas9 technology is not yet used as a single therapy to treat tumors but it can be combined with traditional treatment strategies to provide personalized gene therapy for patients. The combination with chemotherapy, radiation and immunotherapy has been proven to be a powerful means of screening, identifying, validating and correcting tumor targets. Recently, CRISPR/Cas9 technology and CAR T-cell therapies have been integrated to open novel opportunities for the production of more efficient CAR T-cells for all patients. GMP-compatible equipment and reagents are already available for several clinical-grade systems at present, creating the basis and framework for the accelerated development of novel treatment methods. Conclusion: Here we will provide a comprehensive collection of the actual GMP-grade CRISPR/Cas9-mediated approaches used to support cancer therapy highlighting how this technology is opening new opportunities for treating tumors. [ABSTRACT FROM AUTHOR]

• The tendon circadian clock regulates the synthesis of collagen i and the appearance and disappearance of small-diameter collagen fibrils in the matrix. • Matrix metalloproteinase 14 expression is also circadian rhythmic. • Knockout of Mmp14 in fibroblasts led to an accumulation of small-diameter fibrils. • There were major changes in the tendon matrisome, including increase in collagen crosslinking enzymes. • Loss of Mmp14 significantly dampened the circadian rhythm. The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts. [ABSTRACT FROM AUTHOR]

Several studies have introduced CRISPR-Cas9 as a prominent genome-editing technology. CRISPR was first established as a bacterial immune system and was later founded as a new genome-editing technology by Jennifer Doudna and colleagues around the turn of the decade. CRISPR is preceded by zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs); however, it improves upon these previous genome-editing designs by using RNA as its binding domain and by already containing nucleases—reducing the need to intentionally engineer these domains into the technology. Complementarity between the different RNAs (tracrRNA and crRNA) and protein aspects (Cas systems) of CRISPR act in conjunction to both efficiently assemble and confer its intended purpose. After extensive use in mammalian cell cultures and various model organisms, the CRISPR-Cas system has subsequently been used to mediate several genetic diseases such as sickle cell anemia and some cancers in humans. CRISPR-Cas9’s potential to mediate all other genetic mutations induced by sequence polymorphisms remains promising.

Scallops have become an important aquaculture species in China because they contain high-quality protein, and scallops are important health food that combines multiple effects and high economic benefits. However, scallop aquaculture is perennially threatened by various pathogenic Vibrio species, leading to great economic losses. We obtained a strain of pathogenic bacteria, identified as Vibrio alginolyticus, from the diseased Azumapecten farreri in the scallop farming area of Huangdao District in 2018, and V. alginolyticus is one of the major shellfish pathogens. We showed that V. alginolyticus was isolated and identified as a pathogen in A. farreri for the first time. In this study, we evaluated its morphology and performed a phylogenetic analysis based on 16S rRNA gene sequencing. In addition, we performed a preliminary analysis of its pathogenic mechanisms. The Hfq protein in V. alginolyticus is an important RNA-binding protein in the quorum-sensing system that not only affects the sensitivity of Vibrio to environmental stress but also regulates a variety of functions, such as cell membrane formation, motility, and virulence towards the host. However, its effect on the pathogenesis of V. alginolyticus to A. farreri is unclear. To further investigate the pathogenic mechanism of the Hfq protein in V. alginolyticus to A. farreri, we used the CRISPR-Cas9 system to target and deplete the hfq gene fragment in V. alginolyticus and obtained the mutant strain V. ΔHfq−. We found that the peripheral flagellum of the mutant strain was lost, which reduced the motility of V. alginolyticus. Therefore, the deletion of target genes by the CRISPR/Cas9 genome editing system confirmed that the Hfq protein played a key role in reducing the ability of V. alginolyticus to infect A. farreri. In conclusion, our current findings provided valuable insights into the healthy culture of scallops. [ABSTRACT FROM AUTHOR]

Study questionDo seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)?Summary answerSP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization.What is known alreadyClinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis.Study design, size, durationThis is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization.Participants/materials, setting, methodseSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs.Main results and the role of chanceSP promoted decidualization both in the absence and presence of steroid hormones (P

Abstract Background The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9. Methods OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2′-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model. Results Approximately 70–90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model. Conclusions Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer.

Plant-parasitic nematodes (PPNs) pose a threat to global food security in both the developed and developing worlds. PPNs cause crop losses worth a total of more than USD 150 billion worldwide. The sedentary root-knot nematodes (RKNs) also cause severe damage to various agricultural crops and establish compatible relationships with a broad range of host plants. This review aims to provide a broad overview of the strategies used to identify the morpho-physiological and molecular events that occur during RKN parasitism. It describes the most current developments in the transcriptomic, proteomic, and metabolomic strategies of nematodes, which are important for understanding compatible interactions of plants and nematodes, and several strategies for enhancing plant resistance against RKNs. We will highlight recent rapid advances in molecular strategies, such as gene–silencing technologies, RNA interference (RNAi), and small interfering RNA (siRNA) effector proteins, that are leading to considerable progress in understanding the mechanism of plant–nematode interactions. We also take into account genetic engineering strategies, such as targeted genome editing techniques, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system, and quantitative trait loci (QTL), to enhance the resistance of plants against nematodes. [ABSTRACT FROM AUTHOR]

Genomics and genome editing promise enormous opportunities for crop improvement and elementary research. Precise modification in the specific targeted location of a genome has profited over the unplanned insertional events which are generally accomplished employing unadventurous means of genetic modifications. The advent of new genome editing procedures viz; zinc finger nucleases (ZFNs), homing endonucleases, transcription activator like effector nucleases (TALENs), Base Editors (BEs), and Primer Editors (PEs) enable molecular scientists to modulate gene expressions or create novel genes with high precision and efficiency. However, all these techniques are exorbitant and tedious since their prerequisites are difficult processes that necessitate protein engineering. Contrary to first generation genome modifying methods, CRISPR/ Cas9 is simple to construct, and clones can hypothetically target several locations in the genome with different guide RNAs. Following the model of the application in crop with the help of the CRISPR/Cas9 module, various customized Cas9 cassettes have been cast off to advance mark discrimination and diminish random cuts. The present study discusses the progression in genome editing apparatuses, and their applications in chickpea crop development, scientific limitations, and future perspectives for biofortifying cytokinin dehydrogenase, nitrate reductase, superoxide dismutase to induce drought resistance, heat tolerance and higher yield in chickpea to encounter global climate change, hunger and nutritional threats. [ABSTRACT FROM AUTHOR]

Genomics and genome editing promise enormous opportunities for crop improvement and elementary research. Precise modification in the specific targeted location of a genome has profited over the unplanned insertional events which are generally accomplished employing unadventurous means of genetic modifications. The advent of new genome editing procedures viz; zinc finger nucleases (ZFNs), homing endonucleases, transcription activator like effector nucleases (TALENs), Base Editors (BEs), and Primer Editors (PEs) enable molecular scientists to modulate gene expressions or create novel genes with high precision and efficiency. However, all these techniques are exorbitant and tedious since their prerequisites are difficult processes that necessitate protein engineering. Contrary to first generation genome modifying methods, CRISPR/Cas9 is simple to construct, and clones can hypothetically target several locations in the genome with different guide RNAs. Following the model of the application in crop with the help of the CRISPR/Cas9 module, various customized Cas9 cassettes have been cast off to advance mark discrimination and diminish random cuts. The present study discusses the progression in genome editing apparatuses, and their applications in chickpea crop development, scientific limitations, and future perspectives for biofortifying cytokinin dehydrogenase, nitrate reductase, superoxide dismutase to induce drought resistance, heat tolerance and higher yield in chickpea to encounter global climate change, hunger and nutritional threats.

Background: The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9. Methods: OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2′-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model. Results: Approximately 70–90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model. Conclusions: Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer. [ABSTRACT FROM AUTHOR]

Rice (Oryza sativa L.) is an important staple food crop worldwide. To meet the growing nutritional requirements of the increasing population in the face of climate change, qualitative and quantitative traits of rice need to be improved. During recent years, genome editing has played a great role in the development of superior varieties of grain crops. Genome editing and speed breeding have improved the accuracy and pace of rice breeding. New breeding technologies including genome editing have been established in rice, expanding the potential for crop improvement. Over a decade, site-directed mutagenesis tools like Zinc Finger Nucleases (ZFN), Transcriptional activator-like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) System were used and have played a great role in rice yield and quality enhancement. In addition, most recently other genome editing techniques like prime editing and base editors have also been used for efficient genome editing in rice. Since rice is an excellent model system for functional studies due to its small genome and close synthetic relationships with other cereal crops, new genome-editing technologies continue to be developed for use in rice. Genomic alteration employing genome editing technologies (GETs) like CRISPR/Cas9 for reverse genetics has opened new avenues in agricultural sciences such as rice yield and grain quality improvement. Currently, CRISPR/Cas9 technology is widely used by researchers for genome editing to achieve the desired biological objectives, because of its simple targeting, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. Over the past few years many genes related to rice grain quality and yield enhancement have been successfully edited via CRISPR/Cas9 technology method to cater to the growing demand for food worldwide. The effectiveness of these methods is being verified by the researchers and crop scientists worldwide. In this review we focus on genome-editing tools for rice improvement to address the progress made and provide examples of genome editing in rice. We also discuss safety concerns and methods for obtaining transgene-free crops.

Abstract Cancer immunotherapy has gained attention as the supreme therapeutic modality for the treatment of various malignancies. Adoptive T-cell therapy (ACT) is one of the most distinctive modalities of this therapeutic approach, which seeks to harness the potential of combating cancer cells by using autologous or allogenic tumor-specific T-cells. However, a plethora of circumstances must be optimized to produce functional, durable, and efficient T-cells. Recently, the potential of ACT has been further realized by the introduction of novel gene-editing platforms such as the CRISPR/Cas9 system; this technique has been utilized to create T-cells furnished with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR) that have precise tumor antigen recognition, minimal side effects and treatment-related toxicities, robust proliferation and cytotoxicity, and nominal exhaustion. Here, we aim to review and categorize the recent breakthroughs of genetically modified TCR/CAR T-cells through CRISPR/Cas9 technology and address the pearls and pitfalls of each method. In addition, we investigate the latest ongoing clinical trials that are applying CRISPR-associated TCR/CAR T-cells for the treatment of cancers.

Plant-parasitic nematodes (PPNs) pose a threat to global food security in both the developed and developing worlds. PPNs cause crop losses worth a total of more than USD 150 billion worldwide. The sedentary root-knot nematodes (RKNs) also cause severe damage to various agricultural crops and establish compatible relationships with a broad range of host plants. This review aims to provide a broad overview of the strategies used to identify the morpho-physiological and molecular events that occur during RKN parasitism. It describes the most current developments in the transcriptomic, proteomic, and metabolomic strategies of nematodes, which are important for understanding compatible interactions of plants and nematodes, and several strategies for enhancing plant resistance against RKNs. We will highlight recent rapid advances in molecular strategies, such as gene–silencing technologies, RNA interference (RNAi), and small interfering RNA (siRNA) effector proteins, that are leading to considerable progress in understanding the mechanism of plant–nematode interactions. We also take into account genetic engineering strategies, such as targeted genome editing techniques, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system, and quantitative trait loci (QTL), to enhance the resistance of plants against nematodes.

Abstract Background β-Thalassemias represent a group of genetic disorders caused by human hemoglobin beta (HBB) gene mutations. The radical curative approach is to correct the mutations causing the disease. CRISPR-CAS9 is a novel gene-editing technology that can be used auspiciously for the treatment of these disorders. The study aimed to investigate the utility of CRISPR-CAS9 for gene modification of hematopoietic stem cells in β-thalassemia with IVS-1-110 mutation. Methods and results We successfully isolated CD34+ cells from peripheral blood of β-thalassemia patients with IVS-1-110 mutation. The cells were transfected with Cas9 endonuclease together with guide RNA to create double-strand breaks and knock out the mutation. The mutation-corrected CD34+ cells were subjected to erythroid differentiation by culturing in complete media containing erythropoietin. Conclusion CRISPR/Cas-9 is an effective tool for gene therapy that will broaden the spectrum of therapy and potentially improve the outcomes of β-thalassemia.

Cancer immunotherapy has gained attention as the supreme therapeutic modality for the treatment of various malignancies. Adoptive T-cell therapy (ACT) is one of the most distinctive modalities of this therapeutic approach, which seeks to harness the potential of combating cancer cells by using autologous or allogenic tumor-specific T-cells. However, a plethora of circumstances must be optimized to produce functional, durable, and efficient T-cells. Recently, the potential of ACT has been further realized by the introduction of novel gene-editing platforms such as the CRISPR/Cas9 system; this technique has been utilized to create T-cells furnished with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR) that have precise tumor antigen recognition, minimal side effects and treatment-related toxicities, robust proliferation and cytotoxicity, and nominal exhaustion. Here, we aim to review and categorize the recent breakthroughs of genetically modified TCR/CAR T-cells through CRISPR/Cas9 technology and address the pearls and pitfalls of each method. In addition, we investigate the latest ongoing clinical trials that are applying CRISPR-associated TCR/CAR T-cells for the treatment of cancers. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

SETDB1 HMTase participates in various cellular processes via epigenetic transcriptional regulation. SETDB1 expression is downregulated by anticancer drug treatment in cancer cells, but we still need to verify the functional significance on SETDB1 downregulation. CRISPR/cas9 is a useful technology for doing a knockout (KO) of a target gene. It is widely used to examine the function of genes. In this study, we prepared SETDB1-KO from A549 human lung cancer cells using the CRISPR/Cas9 system, and we compared molecular changes between the A549 cells and the SETDB1-KO cells. The SETDB1-KO cell proliferation rate was slightly decreased as compared to the A549 cells, but there was no large difference in sensitivity with doxorubicin treatment. Instead, the migration activity and transforming activity were dramatically increased in SETDB-KO cells. Using a western blot analysis and an immunostaining experiment, we confirmed that SETDB1-KO downregulates the expression of E-cadherin and β-catenin. A qPCR and an RT-PCR analysis suggested that SETDB1 transcriptionally regulates E-cadherin and β-catenin. Moreover, E-cadherin expression was also detected in the cytoplasmic region of SETDB1-KO cells, indicating that functional localization of E-cadherin might be changed in SETDB1-KO cells. On the other hand, total levels of STAT3 and Akt were increased in the SETDB1-KO cells, but activation of STAT3 (pSTAT3) was not induced in doxorubicin-treated SETDB1-KO cells. SETDB1 overexpression into SETDB1-KO cells restores the expression of E-cadherin, β-catenin, STAT3, and Akt, suggesting that those proteins are tightly regulated by SETDB1. Collectively, we suggest that complex regulations on E-cadherin, β-catenin, STAT3, and Akt are correlated with the increased migration and transforming activity of SETDB1-KO cells. • SETDB1-KO has no influence on doxorubicin sensitivity but increases migration and transforming activity. • SETDB1-KO downregulates expression of E-cadherin and ꞵ-catenin, whereas upregulates expression of STAT3 and Akt. • SETDB1 overexpression restores the expression of E-cadherin, β-catenin, STAT3, and Akt. • Complex regulations on those proteins are correlated with the increased migration and transforming activity of SETDB1-KO cells. [ABSTRACT FROM AUTHOR]

Purpose: To correct a potentially damaging mutation in haploid human embryonic stem cells. Methods: Exome sequencing was performed on DNA extracted from parthenogenetically derived embryonic stem cell line (pES12). An SLC10A2 gene mutation, which affects bile acid transport, was chosen as mutation of interest in this proof of concept study to attempt correction in human pluripotent haploid cells. Confirmation of the mutation was verified, and guide RNA and a correction template was designed in preparation of performing CRISPR. Haploid cells underwent serial fluorescence activated cell sorting (FACS) with Hoechst 33342 to create an increasingly haploid (1n) enriched culture. Nucleofection was performed on p. 37 and then cells were sorted for 1n DNA content with +GFP to identify the haploid cells that expressed Cas9 tagged with GFP. Results: 104,686 haploid GFP + cells were collected. Cells were cultured, individual colonies picked, and 48 clones were sent for Sanger sequencing. CRIPSR efficiency was 77.1%, with 7/48 (14.6%) clones resulting in a corrected SLC10A2 mutation. Confirmation of persistence of haploid cells was achieved with repeated FACS sorting and centromere quantification. Given the large number of passages and exposure to CRISPR, we also performed analysis of karyotypes and of off-target effects. Cells evaluated were karyotypically normal and there was no evident off target effects. Conclusions: CRISPR/Cas9 can be effectively utilized to edit mutations in haploid human embryonic stem cells. Establishment and maintenance of a haploid cell culture provides a novel way to utilize CRISPR/Cas9 in gene editing, particularly in the study of recessive alleles. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease accounts as the leading cause of mortality worldwide prominently affected by genetic and environmental factors. The disease is characterized by persistent coughing, breathlessness airways inflammation followed by a decrease in forced expiratory volume1 and exacerbations, which affect the quality of life. Determination of genetic, epigenetic, and oxidant biomarkers to evaluate the progression of disease has proved complicated and challenging. Approaches including exome sequencing, genome‐wide association studies, linkage studies, and inheritance and segregation studies played a crucial role in the identification of genes, their pathways and variation in genes. This review highlights multiple approaches for biomarker and gene identification, which can be used for differential diagnosis along with the genome editing tools to study genes associated with the development of disease and models their function. Further, we have discussed the approaches to rectify the abnormal gene functioning of respiratory tissues and various novel gene editing techniques like Zinc finger nucleases (ZFN), transcription activator‐like effector nucleases (TALEN), and clustered regulatory interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9). [ABSTRACT FROM AUTHOR]

A total monosaccharide concentration of 47.0 g/L from 12% (w/v) Gracilaria verrucosa was obtained by hyper thermal acid hydrolysis with 0.2 M HCl at 140°C for 15 min and enzymatic saccharification with CTec2. To improve galactose utilization, we overexpressed two genes, SNR84 and PGM2, in a Saccharomyces cerevisiae CEN-PK2 using CRISPR/Cas-9. The overexpression of both SNR84 and PGM2 improved galactose utilization and ethanol production compared to the overexpression of each gene alone. The overexpression of both SNR84 and PGM2 and of PGM2 and SNR84 singly in S. cerevisiae CEN-PK2 Cas9 produced 20.0, 18.5, and 16.5 g/L ethanol with ethanol yield (YEtOH) values of 0.43, 0.39, and 0.35, respectively. However, S. cerevisiae CEN-PK2 adapted to high concentration of galactose consumed galactose completely and produced 22.0 g/L ethanol at a YEtOH value of 0.47. The overexpression of both SNR84 and PGM2 increased the transcriptional levels of GAL and regulatory genes; however, the transcriptional levels of these genes were lower than those in S. cerevisiae adapted to high galactose concentrations. [ABSTRACT FROM AUTHOR]

The rising interest in human induced pluripotent stem cell (hiPSC)-derived organoid culture has stemmed from the manipulation of various combinations of directed multi-lineage differentiation and morphogenetic processes that mimic organogenesis. Organoids are three-dimensional (3D) structures that are comprised of multiple cell types, self-organized to recapitulate embryonic and tissue development in vitro. This model has been shown to be superior to conventional two-dimensional (2D) cell culture methods in mirroring functionality, architecture, and geometric features of tissues seen in vivo. This review serves to highlight recent advances in the 3D organoid technology for use in modeling complex hereditary diseases, cancer, host-microbe interactions, and possible use in translational and personalized medicine where organoid cultures were used to uncover diagnostic biomarkers for early disease detection via high throughput pharmaceutical screening. In addition, this review also aims to discuss the advantages and shortcomings of utilizing organoids in disease modeling. In summary, studying human diseases using hiPSC-derived organoids may better illustrate the processes involved due to similarities in the architecture and microenvironment present in an organoid, which also allows drug responses to be properly recapitulated in vitro. [ABSTRACT FROM AUTHOR]

Abstract: Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO‐1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1–/– fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO‐1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53‐regulated miR‐34a and 14‐3‐3σ was observed in HO‐1‐deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO‐1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1+/+ and Hmox1–/– iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO‐1‐deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas‐9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2‐lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2+/+ and Nfe2l2–/– iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO‐1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129–142, 2018 [ABSTRACT FROM AUTHOR]

Efficiency of artificial restriction enzymes toward curing HIV has only been separately examined, using differing delivery vehicles. We compared the in vitro transduction and target-mutagenesis efficiency of consortium plasmid and adenoviral vector delivered HIV-1 pol gene targeting zinc finger nuclease (ZFN) with CRISPR/Cas, Custom-ZFN, CRISPR-Cas-9, and plasmids and vectors (murCTSD_pZFN, pGS-U-gRNA, pCMV-Cas-D01A, Ad5-RGD); cell lines (TZM-bl and ACH-2/J-Lat cells); and the latency reversing agents prostratin, suberoylanilide hydroxamic acid, and phorbol myristate acetate. Cell lines were grown in either Dulbecco's modified Eagle's medium or Roswell Park Memorial Institute with the antibiotics kanamycin, zeocin, and efavirenz. Efficiency was assayed by GFP/luciferase activity and/or validated by yeast MEL1 reporter assay, CEL1 restriction fragment assay, and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Ad5-RGD vectors had better transduction efficiency than murCTSD and pGS-U-gRNA/pCMV-Cas-D01A plasmids. CRISPR/Cas9 exhibited better target-mutagenesis efficiency relative to ZFN (delivered by either plasmid or Ad5 vector) based on gel electrophoresis of pol gene amplicons within ACH-2 and J-Lat cells. Ad-5-RGD vectors enhanced target mutagenesis of ZFN, relative to murCTSD_pZFN plasmids, to levels of CRISPR/Cas9 plasmids. Similar reduction of luciferase activity among TZM-bl treated with Ad5-ZFN vectors relative to CRISPR/Cas-9 and murCTSD_pZFN plasmids was observed on challenge with HIV-1. qRT-PCR of HIV-1 pol gene transcripts affirmed that Ad5 (RGD) vectors enhanced target mutagenesis of ZFN. Whereas CRISPR/Cas-9 may possess inherent superior target-mutagenesis efficiency; the efficiency of ZFN (off-target toxicity withstanding) can be enhanced by altering delivery vehicle from plasmid to Ad5 (RGD) vectors. [ABSTRACT FROM AUTHOR]

In a legal 'first', the UK removed a prohibition against modifying embryos in human reproduction, to enable mitochondrial replacement techniques (MRTs), a move the Government distanced from 'germline genetic modification', which it aligned with modifying the nuclear genome. This paper (1) analyzes the uses and meanings of this term in UK/US legal and policy debates; and (2) evaluates related ethical concerns about identity. It shows that, with respect to identity, MRTs and nuclear genome editing techniques such as CRISPR/Cas-9 (now a policy topic), are not as different as has been supposed. While it does not follow that the two should be treated exactly alike, one of the central reasons offered for treating MRTs more permissively than nuclear genetic modification, and for not regarding MRTs as 'germline genetic modification', is thereby in doubt. Identity cannot, by itself, do the work thus far assigned to it, explicitly or otherwise, in law and policy. [ABSTRACT FROM AUTHOR]

Schematic representation of translating HuR targeted therapeutics from bench to bedside for lung cancer. Image created with BioRender.com. [Display omitted] Lung cancer (LC) is often diagnosed at an advanced stage and conventional treatments for disease management have limitations associated with them. Novel therapeutic targets are thus avidly sought for the effective management of LC. RNA binding proteins (RBPs) have been convincingly established as key players in tumorigenesis, and their dysregulation is linked to multiple cancers, including LC. In this context, we review the role of Human antigen R (HuR), an RBP that is overexpressed in LC, and further associated with various aspects of LC tumor growth and response to therapy. Herein, we describe the role of HuR in LC progression and outline the evidences supporting various pharmacologic and biologic approaches for inhibiting HuR expression and function. These approaches, including use of small molecule inhibitors, siRNAs and shRNAs, have demonstrated favorable results in reducing tumor cell growth, invasion and migration, angiogenesis and metastasis. Hence, HuR has significant potential as a key therapeutic target in LC. Use of siRNA-based approaches, however, have certain limitations that prevent their maximal exploitation as cancer therapies. To address this, in the conclusion of this review, we provide a list of nanomedicine-based HuR targeting approaches currently being employed for siRNA and shRNA delivery, and provide a rationale for the immense potential therapeutic benefits offered by nanocarrier-based HuR targeting and its promise for treating patients with LC. [ABSTRACT FROM AUTHOR]

Background: β-Thalassemias represent a group of genetic disorders caused by human hemoglobin beta (HBB) gene mutations. The radical curative approach is to correct the mutations causing the disease. CRISPR-CAS9 is a novel gene-editing technology that can be used auspiciously for the treatment of these disorders. The study aimed to investigate the utility of CRISPR-CAS9 for gene modification of hematopoietic stem cells in β-thalassemia with IVS-1-110 mutation. Methods and results: We successfully isolated CD34+ cells from peripheral blood of β-thalassemia patients with IVS-1-110 mutation. The cells were transfected with Cas9 endonuclease together with guide RNA to create double-strand breaks and knock out the mutation. The mutation-corrected CD34+ cells were subjected to erythroid differentiation by culturing in complete media containing erythropoietin. Conclusion: CRISPR/Cas-9 is an effective tool for gene therapy that will broaden the spectrum of therapy and potentially improve the outcomes of β-thalassemia. [ABSTRACT FROM AUTHOR]