CRISPR-Cas9 Gene Editing System Research Articles

The role of sphingosine 1-phosphate receptor 2 on interleukin (IL)-4/IL-13-induced STAT6 phosphorylation in phorbol 12-myristate 13-acetate-stimulated THP-1 cells<i> </i>

Previously we found that in bleomycin-administered sphingosine 1-phosphate receptor 2 (S1P2) knockout mice, phosphorylation of STAT6, a transcription factor which is activated downstream of interleukin (IL)-4/IL-13, in broncho-alveolar lavage fluids cells was diminished compared with wild-type mice (PLoS One, 2018). In this study, we examined the role of S1P2 on STAT6 activation using THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) as a model for macrophages (MΦs). Quantitative PCR analysis showed that THP-1 cells expresses S1P2. We examined the responses of PMA-treated THP-1 MΦs to chronic stimulation with IL-4/IL-13 in the presence of S1P. IL-4/IL-13 stimulation induced a strong increase in STAT6 phosphorylation in PMA-treated THP-1 MΦs. The major signaling pathway of S1P2 is a Rho–Rho kinase pathway. Pretreatment of PMA-treated THP-1 MΦs with the S1P2 antagonist JTE-013 and the Rho kinase inhibitor Y27632 inhibited IL-4/IL-13–induced increase in STAT6 phosphorylation. We established S1P2knockout THP-1 cells using CRISPR-Cas9 gene editing system. PMA-treated S1P2knockout THP-1 MΦs showed a lower extent of STAT6 phosphorylation in response to IL-4/IL-13 stimulation than PMA-treated wild-type THP-1 MΦs. S1P2 knockout markedly attenuated IL-4/IL-13-induced increase in the mRNA expression of the M2 marker arginase 1. These results suggested that S1P2/Rho kinase pathway is necessary for full activation of STAT6 by IL-4/IL-13 in MΦs. Recently, Th2-type cytokines IL-4/IL-13 have been reported to be important in the pathogenesis of allergic diseases such as bronchial asthma and atopic dermatitis. It is expected that S1P2 antagonist and Rho kinase inhibitor inhibit Th2-type immune response by IL-4/IL-13, thereby suppressing allergic diseases.

Exploiting the CRISPR-Cas9 gene-editing system for human cancers and immunotherapy.

The discovery of clustered regularly interspaced short palindromic repeats and CRISPR‐associated protein 9 (CRISPR‐Cas9) technology has brought advances in the genetic manipulation of eukaryotic cells, which has revolutionised cancer research and treatment options. It is increasingly being used in cancer immunotherapy, including adoptive T and natural killer (NK) cell transfer, secretion of antibodies, cytokine stimulation and overcoming immune checkpoints. CRISPR‐Cas9 technology is used in autologous T cells and NK cells to express various innovative antigen designs and combinations of chimeric antigen receptors (CARs) targeted at specific antigens for haematological and solid tumors. Additionally, advanced engineering in immune cells to enhance their sensing circuits with sophisticated functionality is now possible. Intensive research on the CRISPR‐Cas9 system has provided scientists with the ability to overcome the hostile tumor microenvironment and generate more products for future clinical use, especially off‐the‐shelf, universal cellular products, bringing exciting milestones for immunotherapy. This review discussed the application and challenges of CRISPR technology in cancer research and immunotherapy, its advances and prospects for promoting new cell‐based therapeutic beyond immune oncology.

27. The Membrane Antimicrobial Peptide Defense (MadRS) System Orchestrates Resistance Against Antibiotics and Host Innate Immune Peptides in enterococcus Faecalis

BackgroundEnterococci have evolved a network of two component-signaling systems (TCS) to evade killing by cationic antimicrobial peptides (AP) of the host innate immune response. Activation of these pathways can also lead to cross-resistance to AP-like antibiotics such as daptomycin (DAP), complicating treatment of severe enterococcal infections. Our previous work implicated the MadRS (formerly YxdJK) TCS in resistance to DAP. In this work, we show that the MadRS system regulates a network of membrane defense proteins that provide targeted resistance to killing by LL-37.MethodsWe performed a global transcriptional analysis comparing wild type (WT) E. faecalis OG1RF and an OG1RF derivative possessing a mutated allele of the madS gene that activates the MadRS system (OG1RFmadSA202E). Selected genes identified in this analysis were knocked out using a CRISPR-cas9 gene editing system and confirmed by sequencing. Antibiotic minimum inhibitory concentrations (MIC) were determined using gradient diffusion strips. LL-37 killing assays were performed in triplicate in RPMI+5% LB broth. Caenorhabditis elegans assays were performed in triplicate, and representative results are shown.ResultsAnalysis of the MadRS regulon revealed a significant upregulation of a network of gene targets, including madLM (formerly yxdLM, involved in bacitracin resistance), madEFG, the dlt operon, salA, and EF2211 (Fig 1). The madSA202E allele was associated with increased bacterial survival on exposure to LL-37, while the madEFG deletion mutant was more susceptible to killing (Fig 2A). Deletion of madLM decreased bacitracin MICs, but did not alter survival on exposure to LL-37. In C. elegans, inactivation of the MadRS system by deletion of the madR response regulator resulted in attenuation as compared to WT OG1RF. The effect was reversed by complementing with wild-type madR on a plasmid (Fig 2B). The attenuation effect of the mutant was abolished in pmk-1 knockout worms, which lack the ability to produce innate immune peptides (Fig 2C).Figure 1 Figure 2ConclusionThe MadRS system plays an important role in mediating resistance to AP both in vitro and in vivo. The novel ABC-transporter MadEFG is likely to play a major role in preventing enterococcal killing by the human cathelicidin LL-37. Disclosures William R. Miller, MD, Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support)Shionogi (Advisor or Review Panel member) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)

Potentiate Immune Activation and Function By Targeting Inhibitor of Apoptosis Proteins (IAPs) in Relapse/Refractory DLBCL

Background: The prognosis of relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL) that had progressed after or are ineligible for high dose chemotherapy and autologous stem cell support (HDC-ASCS) is extremely poor (median overall survival of 6.3 months). Cellular based immunotherapy with Chimeric Antigen Receptor T-cell against CD19 (CART-19) has proven high effective in r/r DLBCL with an overall response rate of 52-83% and an overall survival rate of 40%. While r/r DLBCL benefits from CART-19 therapy, a significant number of progress after initial response stresses the need to improve the efficacy of this novel approach. A significant amount of work is being done in improving the manufacturing or activation of CART cells. However, there is few data on lymphoma associated factors that could influence response to CART-19 therapy. Previously we reported an imbalance and dysfunction of pro- and anti-apoptotic proteins, including Bak/Bax, Mcl-1/BCLxL/Survivin and upregulation of inhibitor of apoptosis (IAP) family proteins in rituximab+chemotherapy (R-chemo) resistant DLBCL. Targeting IAPs by small inhibitor LCL161 in lymphoma cells significantly overcame chemotherapy resistance in vitro and in vivo. LCL161 potentiated CART-19 cell cytotoxicity mediated in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma pre-clinical models. Here, we investigate the mechanism of action to enhance immune activation by targeting IAPs in r/r DLBCL. Methods: Primary T-cells were isolated and activated from peripheral blood apheresis samples from consenting adults (healthy human donors or patients with DLBCL) using Pan T-Cell Isolation Kit (MACS) and activated by T-cell Dynabeads human activator CD3/CD28 kit. Cells were expanded in RPMI media supplemented with IL2 for 2 weeks. At day 14, cells phenotype analysis was performed (CD3, CD4, CD8) by flow cytometry. T-cell number was calculated by trypan blue exclusion assay and proliferation was measured by presto blue assay after exposure to LCL161 or control for 48hrs. After exposure, CD4 and CD8 ratios were determined by cellular surface staining. T-cell function assay was determined by intracellular staining with perforin and granzyme B. Regulatory T-cells were determined as CD25+ and FOXP3+ staining cells. T-cell cytolytic activity was accessed by 51Cr release assay using 51Cr labeled rituximab-chemotherapy sensitive (Raji and RL cells) or resistant (Raji 4RH and RL 4RH) cell lines as target cells (effector/target ratio 10/1). To confirm the role of IAP proteins in the effects of LCL161 in T-cell activation, we conducted cytotoxicity assays using XIAP knock out lymphoma cell lines as target cells. Stable XIAP knock out cell lines were generated by CRISPR-Cas9 gene editing system. Lentiviral Cas9 nuclease particles were transduced into Raji and Raji 4RH cells followed by lentiviral XIAP sgRNA transduction. Lentiviral sgRNA non-targeting control was also used as negative control. Result: In vitro exposure of T-cells to LCL161 did not affect cell viability. On the other hand, it led to an increase in CD8+ T-cells and in the CD8:CD4 ratio. In addition, the percentage of regulatory T cells (CD25+ and FOXP3+) was reduced following LCL161 exposure. Using the T-cell function assay, we found that perforin and granzyme B inside T-cell was significantly increased post LCL161 exposure. The presence of LCL161 significantly improved the T-cell killing activity towards rituximab-chemotherapy sensitive and more importantly resistant cell lines. XIAP knock out in target cells resulted in abrogation of the enhance cytotoxicity exhibited by T-cells exposed to LCL161. Conclusion: Our data suggest that besides its previously documented direct anti-tumor activity, LCL161 has dual functions modulating immune cells function. LCL161 improve T-cell function by increasing the number of CD8+ T-cells and intracellular levels of granzyme B and perforin leading to an increase in T-cell cytotoxicity. In addition, LCL161 decreases the number of T-reg cells further enhancing immune activation. Taking together, targeting IAPs would be an attractive approach that potentiate current CART-19 immunotherapy in the clinical setting. Disclosures Hernandez-Ilizaliturri: Astra Zeneca: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Seattle Genetics: Consultancy; Epyzome: Consultancy; Pharmacyclics: Consultancy; Amgen: Consultancy; Gilead: Consultancy.

Knockout of STK10 promotes the migration and invasion of cervical cancer cells.

BackgroundSerine threonine kinase 10 (STK10) is an ERM kinase involved in the activation of ERM proteins and plays an essential role in the aggregation and adhesion of lymphocytes. STK10 is expressed in about 17 cancer types, including cervical cancer. Cervical cancer is the fourth most common cancer that seriously threatens women’s health worldwide. Previous studies have shown that STK10 may affect LFA-1-mediated cell adhesion. Other studies reported a mutation (R634H) of STK10 detected in peripheral T-cell lymphoma. This study aimed to evaluate the functional roles of STK10 in the pathogenesis of cervical cancer.MethodsWe generated STK10 knockout cervical cancer cell lines using the CRISPR-Cas9 gene-editing system, and further analyzed the effects of STK10 deficiency on tumor biological behaviors. The proliferation, apoptosis, migration and invasive activity of these cells were respectively detected by BrdU incorporation, AnnexinV/propidium iodide (PI) staining, wound healing assay and Transwell assays without and with Matrigel. The phosphorylation and expression level of indicated proteins were analyzed by Western blot. The differential expression genes between STK10 knockout and control cells were identified by RNA-seq analysis and further confirmed using qRT-PCR.ResultsOur data revealed that target deletion of STK10 does not affect cell proliferation and apoptosis, but promotes the adhesion, migration, and invasion of cervical cancer cells. Most strikingly, the phosphorylation and expression level of ezrin and other ERM proteins in STK10 knockout cells was comparable with that in the control cells. Further, RNA-seq analysis indicated that the knockout of STK10 resulted in a profound alteration of gene expression in cervical cancer cells.ConclusionsThis is the first study to provide evidence that STK10 executes various physiological functions in addition to phosphorylation of ERM proteins, and plays a vital role in the migration and invasion of cervical cancer cells.

Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies.

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate-mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)-encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

Efficient Editing of an Adenoviral Vector Genome with CRISPR/Cas9.

Immunotherapy based on genetic modification of T cells has played an important role in the treatment of tumors and viral infections. Moreover, adenoviral vectors engineered with improved safety due to their inability to integrate into the host genome have been key in the clinical application of T cell therapy. However, the commonly used adenoviral vector Ad5 exhibits low efficiency of infection of human T cells and the details of the intracellular trafficking pathway of adenoviral vectors in human primary T cells remains unclear. Resolution of these issues will depend on successful modification of the adenoviral vector. To this end, here we describe the successful establishment of a simple and efficient method for editing adenoviral vectors in vitro using the CRISPR-Cas9 gene editing system to target the adenoviral fiber gene.

Abstract 1498: Fibronectin mediates mutation specific interactions between subclonal populations of mutant PIK3CA and HER2 cells in breast cancer

Abstract Breast cancer is the second most common cancer in the world and the most frequently occurring cancer among women. It is a heterogeneous disease and intratumor heterogeneity is one of the main drivers of disease progression, recurrence, therapeutic resistance and metastasis. Herein we have demonstrated that sub clonal populations containing PIK3CA and HER2 mutants exhibit distinct transformative properties accurately modeling a heterogenous tumor in patients. AAV-mediated gene targeting to was used to heterozygously incorporate a PIK3CA (E545K) or a HER2 (L755S or V777L) mutation, separately, into both the MCF-10A and MCF-7 cell lines. Cocultures were carried out by mixing an equal number of cells containing the PIK3CA mutation and cells containing a HER2 mutation into a single culture. Lentiviral transduction was used to tag the cells with either green or red fluorescent proteins to visualize growth patterns in coculture. Cellular fractions in coculture were quantified using droplet digital PCR. Microarray analysis of cocultures identified fibronectin as a significant mediator of growth and fibronectin knockouts were created using the CRISPR Cas9 gene editing system. MCF-10As carrying the PIK3CA mutation are epidermal growth factor (EGF) independent and those carrying the HER2 mutation are highly EGF dependent. We have identified that interactions between the PIK3CA mutants and the HER2 mutants imparts EGF independence to the HER2 mutants. The effect rendered is so potent that the HER2 mutants dominate the coculture kinetics despite the absence of EGF. This distinct transformation was observed both in vitro and in vivo in the tumorigenic MCF-7 cell line. Interestingly, this phenomenon is mutation specific and was not observed in either the MCF-10A and MCF-7 cells containing wildtype HER2 when co-cultured with the PIK3CA mutants. Microarray analysis demonstrated elevated fibronectin, suggesting it may be a mediator of this observation. Competitive inhibition of fibronectin-integrin interaction using synthetic peptides that mimic the RGD binding domain of fibronectin, reduced the growth of HER2 mutants in coculture. Notably, knocking out fibronectin in the PIK3CA mutants reversed the EGF independence observed in the HER2 mutants in both the MCF-10A and MCF -7 cell lines. Our data suggests that mutation specific interactions between mutant PIK3CA and mutant HER2 cells in a genetically heterozygous environment imparts a selective growth advantage to the HER2 mutants. This effect is mediated by an extracellular matrix protein, fibronectin. These interactions could be used to therapeutically target the complications that arise due to tumor heterogeneity. Citation Format: Swathi Karthikeyan, Ian Waters, Lauren Dennison, David Chu, Joshua Donaldson, Dong Ho Shinn, Sarah Croessmann, Ben Ho Park. Fibronectin mediates mutation specific interactions between subclonal populations of mutant PIK3CA and HER2 cells in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1498.

Development of a Self-Restricting CRISPR-Cas9 System to Reduce Off-Target Effects

Development of the CRISPR-Cas9 gene-editing system has given rise to a new era of gene editing with wide applications in biology, medicine, agriculture, and other fields. However, the overexpression of Cas9 nuclease causes off-target effects and may trigger an immune response in vivo. Therefore, we constructed a self-restricting CRISPR-Cas9 system, where the target gene sequence corresponding to the guide RNA (gRNA) is inserted on either end of the Cas9 promoter. When double-strand breaks (DSBs) are induced in the target gene sequence, the Cas9 promoter is cut off and transcription ceases. With this system, expression of Cas9 protein at 60 h after transfection is only 10% that of the wild-type system, with about 70% promoter deletion efficiency. The target site editing efficiency and homologous recombination efficiency of the self-restricting system remain at about 50% and 30%, respectively, while the frequency of off-target indel formation decreased by 76.7%. Further, the number of indel types was also reduced from 13 to 2. Because this system does not include additional gRNA sequences, the possibility of introducing new off-target mutations is decreased. Importantly, this system is composed of a single plasmid, which could potentially be easily introduced in vivo using a viral vector or nanoparticles.

M1 Macrophages Promote TRAIL Expression in Adipose Tissue-Derived Stem Cells, Which Suppresses Colitis-Associated Colon Cancer by Increasing Apoptosis of CD133+ Cancer Stem Cells and Decreasing M2 Macrophage Population.

We have previously reported that adipose tissue-derived stem cells (ASCs) cultured at high cell density can induce cancer cell death through the expression of type I interferons and tumor necrosis factor (TNF)-related apoptosis-inducing ligands (TRAIL). Here, we investigated whether TRAIL-expressing ASCs induced by M1 macrophages can alleviate colitis-associated cancer in an azoxymethane (AOM)/dextran sodium sulfate (DSS) animal model. M1 macrophages significantly increased the TRAIL expression in ASCs, which induced the apoptosis of LoVo cells in a TRAIL-dependent manner. However, CD133knockout LoVo cells, generated using the CRISPR-Cas9 gene-editing system, were resistant to TRAIL. In the AOM/DSS-induced colitis-associated cancer model, the intraperitoneal transplantation of TRAIL-expressing ASCs significantly suppressed colon cancer development. Moreover, immunohistochemical staining revealed a low CD133 expression in tumors from the AOM/DSS + ASCs group when compared with tumors from the untreated group. Additionally, the ASC treatment selectively reduced the number of M2 macrophages in tumoral (45.7 ± 4.2) and non-tumoral mucosa (30.3 ± 1.5) in AOM/DSS + ASCs-treated animals relative to those in the untreated group (tumor 71.7 ± 11.2, non-tumor 94.3 ± 12.5; p < 0.001). Thus, TRAIL-expressing ASCs are promising agents for anti-tumor therapy, particularly to alleviate colon cancer by inducing the apoptosis of CD133+ cancer stem cells and decreasing the M2 macrophage population.

Reconstitution of TCA cycle involving l-isoleucine dioxygenase for hydroxylation of l-isoleucine in Escherichia coli using CRISPR-Cas9.

l-isoleucine dioxygenase (IDO) is an Fe (II)/α-ketoglutarate (α-KG)-dependent dioxygenase that specifically converts l-isoleucine (l-Ile) to (2S, 3R, 4S)-4-hydroxyisoleucine (4-HIL). 4-HIL is an important drug for the treatment and prevention of type 1 and type 2 diabetes but the yields using current methods are low. In this study, the CRISPR-Cas9 gene editing system was used to knockout sucAB and aceAK gene in the TCA cycle pathway of Escherichia coli (E. coli). For single-gene knockout, the whole process took approximately 7days. However, the manipulation time was reduced by 2days for each round of gene modification for multigene editing. Using the genome-edited recombinant strain E. coli BL21(DE3) ΔsucABΔaceAK/pET-28a(+)-ido (2Δ-ido), the bioconversion ratio of L-Ile to 4-HIL was enhanced by about 15% compared to E. coli BL21(DE3)/pET-28a(+)-ido [BL21(DE3)-ido] strain. The CRISPR-Cas9 editing strategy has the potential in modifying multiple genes more rapidly and in optimizing strains for industrial production.

Heterozygous p53-R280T Mutation Enhances the Oncogenicity of NPC Cells Through Activating PI3K-Akt Signaling Pathway

A heterozygous point mutation of p53 gene at codon 280 from AGA to ACA (R280T) frequently occurs in nasopharyngeal carcinoma (NPC) cell lines, and about 10% NPC tissues. However, the role of this mutation in the pathogenesis of NPC remains unclear. In this study, we generated p53 knockout (KO) NPC cell lines from CNE2 cells carrying heterozygous p53 R280T (p53-R280T) mutation and C666-1 cells carrying wild-type p53 by CRISPR-Cas9 gene editing system, and found that KO of heterozygous p53-R280T significantly decreased NPC cell proliferation and increased NPC cell apoptosis, whereas KO of wild-type p53 had opposite effects on NPC cell proliferation and apoptosis. Moreover, KO of heterozygous p53-R280T inhibited the anchorage-independent growth and in vivo tumorigenicity of NPC cells. mRNA sequencing of heterozygous p53-R280T KO and control CNE2 cells revealed that heterozygous p53-R280T mutation activated PI3K-Akt signaling pathway. Moreover, blocking of PI3K-Akt signaling pathway abolished heterozygous p53-R280T mutation-promoting NPC cell proliferation and survival. Our data indicate that p53 with heterozygous R280T mutation functions as an oncogene, and promotes the oncogenicity of NPC cells by activating PI3K-Akt signaling pathway.

ATRX/EZH2 complex epigenetically regulates FADD/PARP1 axis, contributing to TMZ resistance in glioma.

Rationale: Glioma is the most common primary malignant brain tumor in adults. Chemoresistance of temozolomide (TMZ), the first-line chemotherapeutic agent, is a major issue in the management of patients with glioma. Alterations of alpha thalassemia/mental retardation syndrome X-linked (ATRX) gene constitute one of the most prevalent genetic abnormalities in gliomas. Therefore, elucidation of the role of ATRX contributing to TMZ resistance in glioma is urgently needed.Methods: We performed the bioinformatics analysis of gene expression, and DNA methylation profiling, as well as RNA and ChIP-seq data sets. CRISPR-Cas9 gene editing system was used to achieve the ATRX knockout in TMZ resistant cells. In vitro and in vivo experiments were carried out to investigate the role of ATRX contributing to TMZ resistance in glioma.Results: We found that ATRX expression was upregulated via DNA demethylation mediated by STAT5b/TET2 complex and strengthened DNA damage repair by stabilizing PARP1 protein in TMZ resistant cells. ATRX elicited PARP1 stabilization by the down-regulating of FADD expression via the H3K27me3 enrichment, which was dependent on ATRX/EZH2 complex in TMZ resistant cells. Magnetic resonance imaging (MRI) revealed that the PARP inhibitor together with TMZ inhibited glioma growth in ATRX wild type TMZ resistant intracranial xenograft models.Conclusions: The present study further illustrated the novel mechanism of the ATRX/PARP1 axis contributing to TMZ resistance. Our results provided substantial new evidence that PARP inhibitor might be a potential adjuvant agent in overcoming ATRX mediated TMZ resistance in glioma.

COMP-02. MINING-GUIDED MACHINE LEARNING ANALYSES SUPPORTS GRASPING THE LATEST TRENDS ON NEURO-ONCOLOGY

Abstract The systems that can objectively predict the future trends of a particular research field are always anticipated while conducting medical research. Such systems also provide a considerable aid to researchers while determining and acquiring appropriate research budgets. This study intended to establish a novel and versatile algorithm that can predict the latest trends in neuro-oncology. Seventy-nine neuro-oncological research fields were selected using computational sorting methods, such as text-mining analyses, along with 30 journals that represent the recent trends in the neuro-oncology field. Further, the annual impact (AI) for each year with respect to each journal and field (number of articles published in the journal × the impact factor of the journal) was calculated as a novel concept. Subsequently, the AI index (AII) for the year was defined as the sum of the AIs for the aforementioned 30 journals. With respect to the aforementioned neuro-oncological research fields, the AII trends from 2008 to 2017 were subjected to machine learning predicting analyses. The prediction accuracy of the latest trends in neuro-oncology was validated using actual data obtained from previous studies. In particular, the linear prediction model achieved a relatively good accuracy. The most notable and latest predicted fields in neuro-oncology included some interesting emerging fields, such as microenvironment and anti-mitosis, as well as the already renowned fields, such as immunology and epigenetics. Furthermore, we retrospectively attempted an analysis of the fields different from neuro-oncology. Interestingly, as of 2008, the future emergence of the CRISPR-Cas9 gene editing system has been predicted using this system. Overall, the presented algorithm displays potential to be an effective and versatile tool for the prediction of future trends in a particular medical field.

Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean

Small guide RNA (sgRNA) is an important component of the CRISPR/Cas9 system. The gene editing efficiency of the CRISPR/Cas9 system could be enhanced by using highly active U6 promoters to drive the expression of sgRNA. Therefore, we constructed various expression vectors based on the 11 GmU6 promoters predicted and cloned in the whole soybean genome. The expression of truncated GUS driven by 11 GmU6 promoters was tested in hairy roots and by Arabidopsis thaliana transformation. The results indicated that higher transcriptional levels were driven by 5 GmU6 promoters (GmU6-4, GmU6-7, GmU6-8, GmU6-10 and GmU6-11) in both soybean hairy roots and Arabidopsis thaliana. In addition, three genes, Glyma03g36470, Glyma14g04180 and Glyma06g136900, were selected as targets to detect the transcriptional levels of multiple GmU6 promoters. Mutations in these three genes were detected in soybean hairy roots after Agrobacterium rhizogenes infection, indicating efficient target gene editing, including nucleotide insertion, deletion, and substitution. Mutation efficiencies differed among the 11 GmU6 promoters, ranging from 2.8% to 20.6%, and markedly higher efficiencies were obtained with all three genes using the GmU6-8 (20.3%) and GmU6-10 (20.6%) promoters. These two GmU6 promoters also showed higher ability to drive truncated GUS transcription in both soybean hairy roots and transformed Arabidopsis thaliana. These results will help to construct an efficient CRISPR-Cas9 gene editing system and promote the application of the CRISPR-Cas9 genome editing system in soybean molecular breeding.

A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing.

Delivery technologies for the CRISPR-Cas9 gene editing system often require viral vectors, which pose safety concerns for therapeutic genome editing1. Alternatively, cationic liposomal components or polymers can be used to encapsulate multiple CRISPR components into large particles (typically >100 nm diameter); however, such systems are limited by variability in loading of the cargo. Here, we report the design of customizable synthetic nanoparticles for the delivery of Cas9 nuclease and a single-guide RNA (sgRNA), enabling controlled stoichiometry of CRISPR components and limiting possible safety concerns in vivo. We describe the synthesis of a thin glutathione (GSH)-cleavable covalently-crosslinked polymer coating, called a nanocapsule (NC), around a pre-assembled ribonucleoprotein (RNP) complex between a Cas9 nuclease and a sgRNA. The NC is synthesized by acrylate-based polymerization, has a hydrodynamic diameter of 25 nm, and can be customized via facile surface modification. NCs efficiently generate targeted gene edits in vitro without any apparent cytotoxicity. Furthermore, NCs produce robust gene editing in vivo in murine retinal pigment epithelium (RPE) tissue and skeletal muscle following local administration. This customizable NC nanoplatform efficiently delivers CRISPR RNP complexes for in vitro and in vivo somatic gene editing.

Exosome-mediated CRISPR/Cas9 system targets to cut the intercellular transmission function of hepatitis B virus genome

Objective: To determine whether single-stranded guided RNA (gRNA) and Cas9 protein exist in the exosome secreted by cells transfected with CRISPR/Cas9 expression plasmid. Furthermore, to explore whether CRISPR/Cas9 system can edit target genes of peripheral cells through the intercellular transmission of exosomes. Methods: (1) The CRISPR/Cas9 expression plasmid was transfected into HuH7 cells. The supernatant was collected and the exosomes were concentrated and purified by differential centrifugation. The morphology and particle size of exosomes were determined by electronic microscopy and Malvern laser scatter granulometry. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the levels of gRNA and Cas9 protein. (2) HuH7 cells transfected with pBB4.5 1.2×HBV and HBV specific CRISPR/Cas9 expression plasmids were co-cultured. After 2 days, HBV DNA was extracted and sequenced by PCR. Results: There were not only full-length gRNA and Cas9 protein in the exosomes of Huh7 cells transfected with CRISPR / Cas9 expression plasmid. In addition, gene-editing functions were delivered between the cells to achieve the destruction of HBV genome of surrounding cells. Conclusion: The CRISPR-Cas9 gene-editing system has the potential to deliver exosomes between cells via carrying functional gRNA and Cas9 proteins. This phenomenon hints that we are in the process of using the CRISPR/Cas9 system for gene therapy. Therefore, it is necessary to consider the potential effects of exosomes-carried gRNA and Cas9 proteins on the surrounding cells of the distal tubules.

CRISPR-Cas9 mediated CD133 knockout inhibits colon cancer invasion through reduced epithelial-mesenchymal transition.

We previously reported that CD133, as a putative cancer stem cell marker, plays an important role in cell proliferation and invasion in colon cancer. To understand the role of CD133 expression in colon cancer, we evaluated the inhibitory effect of CD133 in colon cancer cells. In this study, we generated CD133knockout colon cancer cells (LoVo) using the CRISPR-Cas9 gene editing system. CD133+ colon cancer cells (LoVo) were infected with the lentiviral vector carrying CD133 gRNA and purified cell by culturing single cell colonies. CD133knockout cells was validated by western blot and flow cytometry analysis. In functional study, we observed a significant reduction in cell proliferation and colony formation in CRISPR-Cas9 mediated CD133 knockout cells in compare with control (P < 0.001). We also found the anticancer effect of stattic was dependent on CD133 expression in colon cancer cells. Although CD133knockout cells could not completely block the tumorigenic property, they showed remarkable inhibitory effects on the ability of cell migration and invasion (P < 0.001). In addition, we examined the epithelial mesenchymal transition (EMT)-related protein expression by western blot. The result clearly showed a loss of vimentin expression in CD133knockout cells. Therefore, CRISPR-Cas9 mediated CD133knockout can be an effective treatment modality for CD133+ colon cancer through reducing the characteristics of cancer stem cells.

Application of CRISPR-Cas9 gene editing to treat HBV

An estimated 291 million people worldwide are living with hepatitis B virus (HBV) infection. Patients with chronic HBV infection are at an increased risk of developing liver cirrhosis and hepatocellular carcinoma (HCC), and about 1 million people died of hepatitis B and its related complications every year. Therefore, HBV represents a significant challenge to public health. The current HBV treatment regime mainly relies on nucleoside (acid) analogs (NUCs) and interferons (IFNs). NUCs inhibit the synthesis of viral DNA from genomic RNA by targeting the multifunctional reverse transcriptase. IFNs acts through both direct antiviral and indirect immunomodulatory effects. Due to the presence of covalently closed circular DNA (cccDNA), the template of HBV transcription, which are highly stable in the nucleus of host cells and difficult to be eliminated by licensed therapies, chronic HBV infection can’t be cured virologically. Consistent efforts have to be made to develop a treatment regime that directly targets persistent cccDNA, inactivates or eliminates it to achieve a totally functional cure for chronic hepatitis B. The clustered regularly interspaced palindromic repeats (CRISPR)-Cas system is an acquired immune system found in prokaryotes. With the development of related research, the CRISPR-Cas9 gene editing system is rapidly becoming an efficient and potentially applicable therapeutic tool. The CRISPR-Cas9 system that specifically targets the conserved regions of the HBV genome, not only effectively inhibits viral replication but also is much simpler and more flexible than other gene editing tools, making it a very attractive treatment option. Numerous experimental studies have shown that the CRISPR-Cas9 system is able to disrupt free cccDNA, excise integrated HBV DNA, reduce pre-genomic RNA and a variety of viral proteins in vitro and in vivo . Furthermore, there are no detectable off-target effects and cytotoxic effects in these relevant anti-HBV studies. However, the feasibility and safety of the CRISPR-Cas9 system still face many challenges in clinical applications, including setting up a more ideal animal model, avoiding unintended off-target effects, achieving high efficiency and low immunogenicity in vivo delivery and establishing a platform of verification that adequately assesses possible risks. While many obstacles need to be overcome, the CRISPR-Cas9 system indeed enhances our ability to change the genome and brings new strategies for treating HBV infection. Besides, the CRISPR-Cas9 systems with better specificity continue to be developed, such as well-designed gRNA and new types or versions of Cas. Viral and no-viral vectors based on in vivo delivery methods for treatment continue to be innovated, and their therapeutic potential will continue to increase. In view of the powerful role on virus suppression of CRISPR-Cas9 technology and the lack of effective means of curing HBV, the CRISPR-Cas9 system still has a bright future in clinical research. This article will summarize the existing CRISPR-Cas9 systems in the study of anti-HBV, and try to explore its possible problems and potential solutions.

Construction of a new isovalerylspiramycin I producing strain by CRISPR-Cas9 system

Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.