Unexpected heterogeneity derived from Cas9 ribonucleoprotein‐introduced clonal cells at the HPRT1 locus

Single‐cell cloning is an essential technique for establishing genome‐edited cell clones mediated by programmable nucleases such as CRISPR‐Cas9. However, residual genome‐editing activity after single‐cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR‐Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single‐cell cloning of Cas9 RNP‐transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single‐cell cloning at 24, 48, 72 hr and 1 week post‐transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high‐resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long‐term incubation before single‐cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single‐cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures.     

T cell receptor‐based cancer immunotherapy: Emerging efficacy and pathways of resistance

Adoptive cell transfer (ACT) using chimeric antigen receptor (CAR)‐modified T cells can induce durable remissions in patients with refractory B‐lymphoid cancers. By contrast, results applying CAR‐modified T cells to solid malignancies have been comparatively modest. Alternative strategies to redirect T cell specificity and cytolytic function are therefore necessary if ACT is to serve a greater role in human cancer treatments. T cell receptors (TCRs) are antigen recognition structures physiologically expressed by all T cells that have complementary, and in some cases superior, properties to CARs. Unlike CARs, TCRs confer recognition to epitopes derived from proteins residing within any subcellular compartment, including the membrane, cytoplasm and nucleus. This enables TCRs to detect a broad universe of targets, such as neoantigens, cancer germline antigens, and viral oncoproteins. Moreover, because TCRs have evolved to efficiently detect and amplify antigenic signals, these receptors respond to epitope densities many fold smaller than required for CAR‐signaling. Herein, we summarize recent clinical data demonstrating that TCR‐based immunotherapies can mediate regression of solid malignancies, including immune‐checkpoint inhibitor refractory cancers. These trials simultaneously highlight emerging mechanisms of TCR resistance. We conclude by discussing how TCR‐based immunotherapies can achieve broader dissemination through innovations in cell manufacturing and non‐viral genome integration techniques.     

Developmental progress of CRISPR/Cas9 and its therapeutic applications for HIV‐1 infection

The CRISPR/Cas9 system has been developed as a powerful tool for targeted gene editing. As a result of technical enhancements in recent years, this technology has become the method of choice for efficiently modifying targeted HIV‐1 genome efficiently as part of HIV therapy. CRISPR can be modified to target specific sequences that Cas9 then cuts. In this article, we outline the development of the CRISPR/Cas9 system. We also show how this technology can be used for the prevention and treatment of HIV‐1 infection. Optimistically, this technology promises to make a significant impact on the fight against HIV‐1 in the future.     

Functional interrogation of Lynch syndrome‐associated MSH2 missense variants via CRISPR‐Cas9 gene editing in human embryonic stem cells

Lynch syndrome (LS) predisposes patients to cancer and is caused by germline mutations in the DNA mismatch repair (MMR) genes. Identifying the deleterious mutation, such as a frameshift or nonsense mutation, is important for confirming an LS diagnosis. However, discovery of a missense variant is often inconclusive. The effects of these variants of uncertain significance (VUS) on disease pathogenesis are unclear, though understanding their impact on protein function can help determine their significance. Laboratory functional studies performed to date have been limited by their artificial nature. We report here an in‐cellulo functional assay in which we engineered site‐specific MSH2 VUS using clustered regularly interspaced short palindromic repeats‐Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild‐type gene. We characterized the impact of the variants on cellular MMR functions including DNA damage response signaling and the repair of DNA microsatellites. We classified the MMR functional capability of eight of 10 VUS providing valuable information for determining their likelihood of being bona fide pathogenic LS variants. This human cell‐based assay system for functional testing of MMR gene VUS will facilitate the identification of high‐risk LS patients.     

Efficient Generation of Gene‐Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9‐Induced Homology‐Directed Repair in Zygotes

Precise genetic mutation of model animals is highly valuable for functional investigation of human mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9)‐induced homology‐directed repair (HDR) is usually used for precise genetic mutation, being limited by the relatively low efficiency compared with that of non‐homologous end joining (NHEJ). Although inhibition of NHEJ was shown to enhance HDR‐derived mutation, in this work, without inhibition of NHEJ, we first generated gene‐modified pigs harboring precise orthologous human mutation (Sox10 c.A325>T) via CRISPR/Cas9‐induced HDR in zygotes using single‐strand oligo DNA (ssODN) as template with an efficiency as high as 80%, indicating that pig zygotes exhibited high activities of HDR relative to NHEJ and were highly amendable to genetic mutation via CIRSPR/Cas9‐induced HDR. Besides, we found a higher concentration of ssODN remarkably reduced HDR‐derived mutation in pig zygotes, suggesting a possible balance for optimal HDR‐derived mutation in zygotes between the excessive accessibility to HDR templates and the activities of HDR relative to NHEJ which appeared to be negatively correlated to ssODN concentration. In addition, the HDR‐derived mutation, as well as those from NHEJ, extensively integrated into various tissues including gonad of founder pig without detected off‐targeting, suggesting CRISPR/Cas9‐induced HDR in zygotes is a reliable approach for precise genetic mutation in pigs.     

Characterization of CRISPR‐Cas systems in Leptospira reveals potential application of CRISPR in genotyping of Leptospira interrogans

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira. However, understanding of the pathogenic mechanism of Leptospira is still elusive due to the limited number of genetic tools available for this microorganism. Currently, the reason for the genetic inaccessibility of Leptospira is still unknown. It is well known that as an acquired immunity of bacteria, Clustered Regularly Interspaced Short Palindromic Repeat‐CRISPR‐associated gene (CRISPR‐Cas) systems can help bacteria against invading mobile genetic elements. In this study, the occurrence and diversity of CRISPR‐Cas systems in 41 genomes of Leptospira strains were investigated. Three subtypes (subtype I‐B, subtype I‐C and subtype I‐E) of CRISPR‐Cas systems were identified in both pathogenic and intermediate Leptospira species but not in saprophytic species. Noteworthy, the majority of pathogenic species harbor two different types of CRISPR‐Cas systems (subtype I‐B and subtype I‐E). Furthermore, Cas2 protein of subtype I‐C in L. interrogans exhibited a metal‐dependent DNase activity in a nonspecific manner. CRISPR spacers in subtype I‐B are highly conserved within the same serovars and hypervariable across different serovars of L. interrogans. Based on the subtype I‐B CRISPR arrays, the serotypes of different L. interrogans strains were easily identified. Investigation of the origin of CRISPR spacers showed that 192 spacers (23.5%) matched to mobile genetic elements, indicating CRISPR‐Cas systems may play an important role in the defense of foreign invading DNA.     

Generation of conditional oncogenic chromosomal translocations using CRISPR–Cas9 genomic editing and homology‐directed repair

Chromosomal rearrangements encoding oncogenic fusion proteins are found in a wide variety of malignancies. The use of programmable nucleases to generate specific double‐strand breaks in endogenous loci, followed by non‐homologous end joining DNA repair, has allowed several of these translocations to be generated as constitutively expressed fusion genes within a cell population. Here, we describe a novel approach that combines CRISPR–Cas9 technology with homology‐directed repair to engineer, capture, and modulate the expression of chromosomal translocation products in a human cell line. We have applied this approach to the genetic modelling of t(11;22)(q24;q12) and t(11;22)(p13;q12), translocation products of the EWSR1 gene and its 3' fusion partners FLI1 and WT1, present in Ewing's sarcoma and desmoplastic small round cell tumour, respectively. Our innovative approach allows for temporal control of the expression of engineered endogenous chromosomal rearrangements, and provides a means to generate models to study tumours driven by fusion genes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Applying gene‐editing technology to elucidate the functional consequence of genetic and epigenetic variation in Alzheimer’s disease

Recent studies have highlighted a potential role of genetic and epigenetic variation in the development of Alzheimer’s disease. Application of the CRISPR‐Cas genome‐editing platform has enabled investigation of the functional impact that Alzheimer’s disease‐associated gene mutations have on gene expression. Moreover, recent advances in the technology have led to the generation of CRISPR‐Cas–based tools that allow for high‐throughput interrogation of different risk variants to elucidate the interplay between genomic regulatory features, epigenetic modifications, and chromatin structure. In this review, we examine the various iterations of the CRISPR‐Cas system and their potential application for exploring the complex interactions and disruptions in gene regulatory circuits that contribute to Alzheimer’s disease.     

Single‐nucleotide polymorphisms associated with pemphigus vulgaris: Potent markers for better treatment and personalized medicine

Pemphigus vulgaris (PV) is a rare autoimmune blistering disorder, which could affect both skin and mucosal surfaces. There is increasing evidence that genetics plays a critical role in PV development, severity and prognosis. Single‐nucleotide polymorphisms (SNPs) are the most common type of genetic variation among people and have been widely evaluated in most diseases. However, there are few studies regarding the roles of SNPs in the PV. Here, we reviewed both pathogenic and protective roles of the SNPs in non‐HLA genes regarding the PV. Among the large number of studied SNPs, it was found that several SNPs in different genes might control the susceptibility of PV, including TNFA (rs361525, rs1800629, rs1800629), IL10 (rs1800871, rs1800896, rs1800871, and rs1800872), IL6 (rs1800795), CTLA4 (rs231775), ICOS (rs10932029), CD86 (rs1129055), DSG3 (rs8085532, rs3911655, rs3848485, rs3794925, rs1466379), ST18 (rs2304365, rs17315309) and TAP2 (rs7454108), probably in a population‐specific manner. Moreover, SNPs in glucocorticoid receptor, also known as nuclear receptor subfamily 3 group C member 1 (NR3C1) gene, including rs11745958, rs17209237, rs33388, rs7701443 as well as rs116855232 at NUDT15, seem to be associated with therapeutic outcomes in PV patients. Additionally, variations in the other genes involved in the drugs' metabolisms, pharmacokinetics and pharmacodynamics such as rs396991 in FCGR3A gene could be used for the prediction of clinical response to drugs and side effects. Taken together, SNPs seem to be valuable tools for better management of PV patients. Further studies need to be conducted to evaluate SNPs in genes that control immune responses and apoptosis.     

The association of interferon‐gamma,interleukin‐4 and interleukin‐17 single‐nucleotide polymorphisms with susceptibility to tuberculosis

Susceptibility to tuberculosis and progression of the disease depend on interactions between the bacterial agent, host immune system, and environmental and genetic factors. In this case‐controlled study, we aimed to determine the role of single‐nucleotide polymorphisms of interferon‐gamma, interleukin‐4 and interleukin‐17 in susceptibility to tuberculosis. Genomic DNA was extracted from peripheral blood samples of patients and controls. The association of single‐nucleotide polymorphisms in interleukin‐4 (?590C/T), interleukin‐17 (?152A/G) and interferon‐gamma (+874T/A) was investigated by polymerase chain reaction (PCR)‐restriction fragment length polymorphism and amplification refractory mutation system‐PCR. A total of 76 tuberculosis patients and 119 healthy individuals were included in this study. The interferon‐gamma (+874T/A) TA genotype was significantly associated with susceptibility to tuberculosis in patients compared to controls (OR = 1.76; 95%CI = 0.84–3.71; p = 0.007), while the interferon‐gamma (+874T/A) TT genotype (OR = 0.51; 95%CI = 0.19–1.36; p = 0.007) had protective effects against tuberculosis and was related to a low risk of tuberculosis development. The difference between allelic and genotypic frequencies of interleukin‐4 (?590C/T) between patients and controls was not significant (p = 0.46). Multivariate logistic regression analysis revealed that the interleukin‐17 (?152A/G) AG genotype (OR = 2.27; 95%CI = 1.19–4.34; p = 0.03) and AA genotype (OR = 1.03; 95%CI = 0.43–2.44; p = 0.03) were significantly different between patients and controls. In conclusion, single‐nucleotide mutations in different cytokine genes may have protective effects or increase the risk of tuberculosis.     

Conjugated Silole and Carbazole Copolymers: Synthesis,Characterization, Single‐Layer Light‐Emitting Diode,and Field Effect Carrier Mobility

Summary: Soluble conjugated random and alternating copolymers (PCz‐PSP) derived from N‐hexyl‐3,6‐carbazole (Cz) and 1,1‐dimethyl‐2,3,4,5‐tetraphenylsilole (PSP) were synthesized by palladium(0)‐catalyzed Suzuki coupling reactions. The feed ratios of Cz to PSP were 95:5, 90:10, 80:20, 70:30, and 50:50. Chemical structures and optoelectronic properties of the copolymers were characterized by ¹H NMR, ¹³C NMR, UV absorption, cyclic voltammetry, photoluminescence, electroluminescence, and field effect transistor. HOMO levels of the copolymers are between −5.15 and −5.34 eV. Single‐layer devices with a configuration of ITO/copolymer/Ba/Al were fabricated and the copolymer with PSP content of 20% displayed the highest external quantum efficiency of 0.77%. Field effect transistors with tantalum pentoxide‐polyacrylonitrile double insulators demonstrated that hole mobilities of the copolymers decreased with their PSP contents, and the hole mobility up to 9.3 × 10⁻⁶ cm² · (V · s)⁻¹ could be achieved.

Synthesis of coplymers derived from 3,6‐carbazole and silole.     

Stimulation‐induced transient changes in neuronal activity,blood flow and N‐acetylaspartate content in rat prefrontal cortex: a chemogenetic fMRS‐BOLD study

Brain activation studies in humans have shown the dynamic nature of neuronal N‐acetylaspartate (NAA) and N‐acetylaspartylglutamate (NAAG) based on changes in their MRS signals in response to stimulation. These studies demonstrated that upon visual stimulation there was a focal increase in cerebral blood flow (CBF) and a decrease in NAA or in the total of NAA and NAAG signals in the visual cortex, and that these changes were reversed upon cessation of stimulation. In the present study we have developed an animal model in order to explore the relationships between brain stimulation, neuronal activity, CBF and NAA. We use “designer receptor exclusively activated by designer drugs” (DREADDs) technology for site‐specific neural activation, a local field potential electrophysiological method for measurement of changes in the rate of neuronal activity, functional MRS for measurement of changes in NAA and a blood oxygenation level‐dependent (BOLD) MR technique for evaluating changes in CBF. We show that stimulation of the rat prefrontal cortex using DREADDs results in the following: (i) an increase in level of neuronal activity; (ii) an increase in BOLD and (iii) a decrease in the NAA signal. These findings show for the first time the tightly coupled relationships between stimulation, neuron activity, CBF and NAA dynamics in brain, and also provide the first demonstration of the novel inverse stimulation–NAA phenomenon in an animal model.     

From Old to New: Polymers from Mono‐α‐Alkylated N‐vinylcaprolactam

N‐vinylcaprolactam (NVCL) is modified via alkylation at the α‐position. The α‐substituents are ethyl (3‐ethyl‐1‐vinylcaprolactam), dodecyl (3‐dodecyl‐1‐vinylcaprolactam), octadecyl (3‐octadecyl‐1‐vinylcaprolactam), 1‐propanol (3‐(3‐hydro­xy‐propyl)‐1‐vinylcaprolactam), and PEG₃ bromide (3‐PEG₃‐bromide‐1‐vinylcaprolactam). These monomers are homo‐ and copolymerized with N‐(4‐methylphenyl)­maleimide by the free radical mechanism. The structures of the obtained polymers are characterized by means of ¹H NMR and IR spectroscopy, gel permeation chromatography (GPC), and by use of differential scanning calorimetry (DSC) (T_g).

     

Brain γ‐aminobutyric acid (GABA) detection in vivo with the J‐editing 1H MRS technique: a comprehensive methodological evaluation of sensitivity enhancement,macromolecule contamination and test–retest reliability

Abnormalities in brain γ‐aminobutyric acid (GABA) have been implicated in various neuropsychiatric and neurological disorders. However, in vivo GABA detection by ¹H MRS presents significant challenges arising from the low brain concentration, overlap by much stronger resonances and contamination by mobile macromolecule (MM) signals. This study addresses these impediments to reliable brain GABA detection with the J‐editing difference technique on a 3‐T MR system in healthy human subjects by: (i) assessing the sensitivity gains attainable with an eight‐channel phased‐array head coil; (ii) determining the magnitude and anatomic variation of the contamination of GABA by MM; and (iii) estimating the test–retest reliability of the measurement of GABA with this method. Sensitivity gains and test–retest reliability were examined in the dorsolateral prefrontal cortex (DLPFC), whereas MM levels were compared across three cortical regions: DLPFC, the medial prefrontal cortex (MPFC) and the occipital cortex (OCC). A three‐fold higher GABA detection sensitivity was attained with the eight‐channel head coil compared with the standard single‐channel head coil in DLPFC. Despite significant anatomical variation in GABA + MM and MM across the three brain regions (p < 0.05), the contribution of MM to GABA + MM was relatively stable across the three voxels, ranging from 41% to 49%, a non‐significant regional variation (p = 0.58). The test–retest reliability of GABA measurement, expressed as either the ratio to voxel tissue water (W) or to total creatine, was found to be very high for both the single‐channel coil and the eight‐channel phased‐array coil. For the eight‐channel coil, for example, Pearson's correlation coefficient of test vs. retest for GABA/W was 0.98 (R² = 0.96, p = 0.0007), the percentage coefficient of variation (CV) was 1.25% and the intraclass correlation coefficient (ICC) was 0.98. Similar reliability was also found for the co‐edited resonance of combined glutamate and glutamine (Glx) for both coils. Copyright © 2016 John Wiley & Sons, Ltd.     

Reversal of type 1 diabetes by a new MHC II‐peptide chimera: “Single‐epitope‐mediated suppression” to stabilize a polyclonal autoimmune T‐cell process

Polyclonality of self‐reactive CD4⁺ T cells is the hallmark of several autoimmune diseases like type 1 diabetes. We have previously reported that a soluble dimeric MHC II‐peptide chimera prevents and reverses type 1 diabetes induced by a monoclonal diabetogenic T‐cell population in double Tg mice [Casares, S. et al., Nat. Immunol. 2002. 3 : 383–391]. Since most of the glutamic acid decarboxylase 65 (GAD65)‐specific CD4⁺ T cells in the NOD mouse are tolerogenic but unable to function in an autoimmune environment, we have activated a silent, monoclonal T‐regulatory cell population (GAD65_217–230‐specific CD4⁺ T cells) using a soluble I‐A/GAD65_217–230/Fcγ2a dimer, and measured the effect on the ongoing polyclonal diabetogenic T‐cell process. Activated GAD65_217–230‐specific T cells and a fraction of the diabetogenic (B_9–23‐specific) T cells were polarized toward the IL‐10‐secreting T‐regulatory type 1‐like function in the pancreas of diabetic NOD mice. More importantly, this led to the reversal of hyperglycemia for more than 2 months post‐therapy in 80% of mice in the context of stabilization of pancreatic insulitis and improved insulin secretion by the β cells. These findings argue for the stabilization of a polyclonal self‐reactive T‐cell process by a single epitope‐mediated bystander suppression. Dimeric MHC class II‐peptide chimeras‐like approach may provide rational grounds for the development of more efficient antigen‐specific therapies in type 1 diabetes.     

Porous Poly(styrene‐co‐divinylbenzene) Neutral Monolith: From Design and Characterization to Reversed‐Phase Capillary Electrochromatography Applications

Macroporous cross‐linked organic polymers, based on styrene and divinylbenzene (PS‐DVB), are prepared as monolithic stationary phases for capillary electrochromatographic applications. The synthetic strategy, which relies on the semi‐interpenetrating polymer network (semi‐IPN) approach, is performed through UV‐initiated free radical copolymerization of the comonomers in the presence of poly(?‐caprolactone) (PCL) within the confines of fused silica capillaries. The characterization of PS‐DVB monoliths is carried out at the different stages of the synthesis using a combination of experimental techniques, thus providing information on the chemical and porous structures. All experimental results evidence the sole role of the PCL oligomer as a porogen.     

Phenotypic spectrum of STRA6 mutations: from Matthew‐Wood syndrome to non‐lethal anophthalmia

Matthew‐Wood, Spear, PDAC or MCOPS9 syndrome are alternative names used to refer to combinations of microphthalmia/anophthalmia, malformative cardiac defects, pulmonary dysgenesis, and diaphragmatic hernia. Recently, mutations in STRA6, encoding a membrane receptor for vitamin A‐bearing plasma retinol binding protein, have been identified in such patients. We performed STRA6 molecular analysis in three fetuses and one child diagnosed with Matthew‐Wood syndrome and in three siblings where two adult living brothers are affected with combinations of clinical anophthalmia, tetralogy of Fallot, and mental retardation. Among these patients, six novel mutations were identified, bringing the current total of known STRA6 mutations to seventeen. We extensively reviewed clinical data pertaining to all twenty‐one reported patients with STRA6 mutations (the seven of this report and fourteen described elsewhere) and discuss additional features that may be part of the syndrome. The clinical spectrum associated with STRA6 deficiency is even more variable than initially described. © 2009 Wiley‐Liss, Inc.