Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives.

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.

Author Correction: Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1-4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.

Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline.

Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a "gene drive," to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an "insertion/deletion" (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of "super-mendelian" inheritance from both male and female mice.

Active genetics comes alive: Exploring the broad applications of CRISPR-based selfish genetic elements (or gene-drives): Exploring the broad applications of CRISPR-based selfish genetic elements (or gene-drives).

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based "active genetic" elements developed in 2015 bypassed the fundamental rules of traditional genetics. Inherited in a super-Mendelian fashion, such selfish genetic entities offered a variety of potential applications including: gene-drives to disseminate gene cassettes carrying desired traits throughout insect populations to control disease vectors or pest species, allelic drives biasing inheritance of preferred allelic variants, neutralizing genetic elements to delete and replace or to halt the spread of gene-drives, split-drives with the core constituent Cas9 endonuclease and guide RNA (gRNA) components inserted at separate genomic locations to accelerate assembly of complex arrays of genetic traits or to gain genetic entry into novel organisms (vertebrates, plants, bacteria), and interhomolog based copying systems in somatic cells to develop tools for treating inherited or infectious diseases. Here, we summarize the substantial advances that have been made on all of these fronts and look forward to the next phase of this rapidly expanding and impactful field.

Mitochondrial diseases in adults.

Mitochondrial medicine is a field that expanded exponentially in the last 30 years. Individually rare, mitochondrial diseases as a whole are probably the most frequent genetic disorder in adults. The complexity of their genotype-phenotype correlation, in terms of penetrance and clinical expressivity, natural history and diagnostic algorithm derives from the dual genetic determination. In fact, in addition to the about 1.500 genes encoding mitochondrial proteins that reside in the nuclear genome (nDNA), we have the 13 proteins encoded by the mitochondrial genome (mtDNA), for which 22 specific tRNAs and 2 rRNAs are also needed. Thus, besides Mendelian genetics, we need to consider all peculiarities of how mtDNA is inherited, maintained and expressed to fully understand the pathogenic mechanisms of these disorders. Yet, from the initial restriction to the narrow field of oxidative phosphorylation dysfunction, the landscape of mitochondrial functions impinging on cellular homeostasis, driving life and death, is impressively enlarged. Finally, from the clinical standpoint, starting from the neuromuscular field, where brain and skeletal muscle were the primary targets of mitochondrial dysfunction as energy-dependent tissues, after three decades virtually any subspecialty of medicine is now involved. We will summarize the key clinical pictures and pathogenic mechanisms of mitochondrial diseases in adults.

The dawn of active genetics.

On December 18, 2014, a yellow female fly quietly emerged from her pupal case. What made her unique was that she had only one parent carrying a mutant allele of this classic recessive locus. Then, one generation later, after mating with a wild-type male, all her offspring displayed the same recessive yellow phenotype. Further analysis of other such yellow females revealed that the construct causing the mutation was converting the opposing chromosome with 95% efficiency. These simple results, seen also in mosquitoes and yeast, open the door to a new era of genetics wherein the laws of traditional Mendelian inheritance can be bypassed for a broad variety of purposes. Here, we consider the implications of this fundamentally new form of "active genetics," its applications for gene drives, reversal and amplification strategies, its potential for contributing to cell and gene therapy strategies, and ethical/biosafety considerations associated with such active genetic elements. Also watch the Video Abstract.

Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.

Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.

Clinical and molecular study in a long-surviving patient with MLASA syndrome due to novel PUS1 mutations.

Myopathy-lactic acidosis-sideroblastic anemia (MLASA) syndrome is a rare autosomal recessive disease. We studied a 43-year-old female presenting since childhood with mild cognitive impairment and sideroblastic anemia. She later developed hepatopathy, cardiomyopathy, and insulin-dependent diabetes. Muscle weakness appeared in adolescence and, at age 43, she was unable to walk. Two novel different mutations in the PUS1 gene were identified: c.487delA (p.I163Lfs*4) and c.884 G>A (p.R295Q). Quantitative analysis of DNA from skeletal muscle biopsies showed a significant increase in mitochondrial DNA (mtDNA) content in the patient compared to controls. Clinical and molecular findings of this patient widen the genotype-phenotype spectrum in MLASA syndrome.

Imaging of mesothelioma of tunica vaginalis testis.

OBJECTIVES: To describe the imaging findings in a series of patients with mesothelioma of the tunica vaginalis testis. METHODS: We reviewed clinical data, imaging findings and follow-up information in a series of 10 pathology-proven cases of mesothelioma (all had US; 2 had MR) of the tunica vaginalis. RESULTS: A variety of patterns could be observed, the most common (5/10) being a hydrocele with parietal, solid and hypervascular vegetations; one patient had a septated hydrocele with hypervascular walls; one had multiple, solid nodules surrounded by a small, physiological quantity of fluid; one a cystic lesion with thick walls and vegetations compressing the testis; two had a solid paratesticular mass. MR showed multiple small nodules on the surface of the tunica vaginalis in one case and diffuse thickening and vegetations in the other one; lesions had low signal intensity on T2-w images and were hypervascular after contrast injection. CONCLUSIONS: A preoperative diagnosis of mesotheliomas presenting as solid paratesticular masses seems very difficult with imaging. On the contrary, the diagnosis must be considered in patients in whom a hydrocele with parietal vegetations is detected, especially if these show high vascularity. KEY POINTS: Mesotheliomas of the tunica vaginalis are rare, often challenging to diagnose preoperatively. Most common finding is a complex hydrocele with hypervascular parietal vegetations. Septated hydrocele, nodules without hydrocele, a thick-walled paratesticular cyst are less common. Preoperative diagnosis may allow aggressive surgical approach and, possibly, a better prognosis.

CEUS Time Intensity Curves in the Differentiation Between Leydig Cell Carcinoma and Seminoma: A Multicenter Study.

PURPOSE: Ultrasound (US) is the main imaging technique in the assessment of testicular masses, as it has proved to be highly accurate in the visualization of these pathologies. Identification of a Leydig cell tumor is essential since the lesion is benign in 90% of cases. The aim of this multicenter study is to assess the effectiveness of contrast-enhanced ultrasound (CEUS) in differentiating Leydig cell tumors from seminoma using qualitative and quantitative features. MATERIALS AND METHODS: From February 2011 to December 2013, 31 patients (mean age: 34 years; range: 25 - 52) were recruited for this prospective study. Three of them were monorchid. Therefore, a total of 59 testicles were assessed. All patients underwent grayscale US, color Doppler ultrasound (CDUS), CEUS and orchiectomy. The paired one-tailed Student's t-test was carried out to differentiate between Leydig cell tumors and seminomas. RESULTS: 31 lesions suspicious for malignancy were hypoechoic on grayscale US while they did not show a typical pattern on CDUS. CEUS qualitative analysis, based on contrast enhancement pattern, during the arterial and venous phases, did not allow discrimination of Leydig cell tumors from seminoma. Quantitative analysis of time-intensity curves (TICs) demonstrated that only three parameters presented statistical significance, i. e. wash-in rate (WiR) p = 0.014, peak enhancement (PE) p = 0.001 and time to peak (TTP) p = 0.003. CONCLUSION: The vascular bed of a Leydig cell tumor is wider and the blood flow velocity is higher than that of a seminoma due to more regular neovascularization. In contrast, a seminoma presents large areas of necrosis due to irregular neovascularization. This explains the different PE and WiR values. Further studies involving larger patient populations are mandatory to confirm these encouraging preliminary results.

Urinary Tract Infection In Young Healthy Women Following Heterosexual Anal Intercourse: Case Reports.

Urinary tract infections (UTIs) are among the most common bacterial infections in outpatient clinical settings globally. Young healthy women are at highest risk of community-acquired UTI. While uncomplicated UTI is not life-threatening, it is associated with high morbidity and treatment costs. The pathogenesis of urinary tract infection in young healthy women is complex. It is influenced by a number of host biological and behavioural factors and virulence of the uropathogen. The infecting uropathogens in community-acquired UTI originate from the fecal flora, E. coli being the most predominant, accounting for 80-90% of these UTIs. Vaginal colonization with uropathogens, a pre-requisite for bladder infection may be facilitated by sexual intercourse, which has been shown to be a strong risk factor and predictor of UTI. While majority of studies have explored the association between heterosexual vaginal intercourse and UTI in healthy young women, the possible association with heterosexual receptive anal intercourse has not received adequate attention despite evidence of high prevalence globally. This paper presents two young healthy married women who had severe UTI following heterosexual anal intercourse and discusses possible association thereof. Understanding the risk factors for UTI and identification of possible predisposing conditions in a particular individual are important in guiding therapeutic approaches and preventive strategies. Cognisant of reportedly high prevalence of various sexual practices including receptive heterosexual anal intercourse and their impact on individuals' health, details on sexual history should always be enquired into in young women presenting with genito-urinary complaints.

Unconsummated marriage in sub-Saharan Africa: case reports.

Unconsummated marriage is a condition where newly married couples are unable to achieve penile-vaginal intercourse for variable periods despite desire and several attempts to do so. Its exact cause(s) is/are unknown, but performance anxiety resulting from or leading to other conditions is reportedly the major etiological factor. It is thought to be more prevalent in traditional and conservative religious communities where premarital sexual exposure is strictly prohibited. Most publications on unconsummated marriage have originated from North America, European and Middle Eastern countries. There have not been any such reports from sub-Saharan Africa, which is home to diverse cultures and traditions regarding premarital sex and marriage. This paper presents a sample of four cases with unconsummated marriage managed by the author in his private clinic based in the city of Nairobi Kenya, over the past five years. Possible etiological factors and management approaches are discussed, with a review of relevant literature.

Influence of night-shift and napping at work on urinary melatonin, 17-ß-estradiol and clock gene expression in pre-menopausal nurses.

Night-workers experience disruption of the sleep-wake cycle and light at night which may increase breast cancer risk by suppressing the nocturnal melatonin surge, resulting in higher levels of circulating estrogens. Night-work may also deregulate peripheral clock genes which have been found to be altered in breast cancer. This study investigated urinary 6-sulfatoxymelatonin (aMT6s), serum 17-beta-estradiol levels in premenopausal shift nurses at the end of the night-shift compared to a control group of daytime nurses. Peripheral clock gene expression in lymphocytes were also investigated. All participants were sampled in the follicular phase of the menstrual cycle. The effect of nurses’ ability to take a short nap during the night-shift was also explored. The shift-work group had significantly lower aMT6s levels than daytime nurses independently of a nap. Night-shift napping significantly influences 17-beta-estradiol levels resulting in higher outcomes in nurses who do not take a nap compared to napping group and daytime workers. Peripheral clock genes expression investigated was not significantly different among the groups. Our findings suggest that shift nurses experience changes in aMT6s levels after a night-shift. Napping habits influence 17-beta-estradiol levels at the end of a night-shift. These findings might be related to the increased cancer risk reported in night-shift workers and suggest that a short nap during night-shifts may exert a positive effect.

Optimisation of radiological protocols for chest imaging using computed radiography and flat-panel X-ray detectors.

PURPOSE: Digital radiography technology has replaced conventional screen-film systems in many hospitals. Despite the different characteristics of new detector materials, frequently, the same radiological protocols previously optimised for screen film are still used with digital equipment without any critical review. This study addressed optimisation of exposure settings for chest examinations with digital systems, considering both image quality and patient dose. MATERIALS AND METHODS: Images acquired with direct digital radiography equipment and a computed radiography system were analysed with specially developed commercial software with a four-alternative forced-choice method: the most promising protocols were then scored by two senior radiologists. RESULTS: Digital technology offers a wide dynamic range and the ability to postprocess images, allowing use of lower tube potentials in chest examinations. The computed radiography system showed both better image quality and lower dose at lower energies (85 kVp and 95 kVp) than those currently used (125 kVp). Direct digital radiography equipment confirmed both its superior image quality and lower dose requirements compared with the storage phosphor plate system. CONCLUSIONS: Generally, lowering tube potentials in chest examinations seems to allow better image quality/effective dose ratio when using digital equipment.

Sexual dysfunction among HIV patients: three case reports and review of literature.

Global efforts in addressing the HIV/AIDS epidemic have focused on preventing new infections, reduction of viral loads through treatment and care and support for the patients. Hardly any attention has been given to their quality of life in particular sexual health and functioning. There is a growing body of literature indicating high prevalence of sexual problems amongst HIV-infected individuals, whose mechanisms remain unclear. This may affect individuals' quality of life, interpersonal relationships and HIV treatment. The sub-Saharan Africa (SSA) region is the epicentre of the HIV epidemic, majority of the patients being young (< 30 years old) and in long-term heterosexual relationships. With increased life expectancy due to expanded access to HAART, the prevalence and potential impact of sexual dysfunction are certain to be significant. There is urgent need for appropriate research on sexual experiences and functioning amongst HIV patients in SSA and appropriate interventions to address them. Current efforts to link HIV/AIDS and sexual and reproductive health and rights (SRHR) and proposals to make SRH services integrated and comprehensive provide are a good starting point. However SRHR policies, strategic plans and programmes should be reviewed to ensure inclusion of sexual health.

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus.

Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test the first CRISPR-based homing gene drive for Culex quinquefasciatus, demonstrating the possibility of using this technology to control Culex mosquitoes. Our results show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes.

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus.

Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test a CRISPR-based homing gene drive for Culex quinquefasciatus, and show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes.

Post-ARDS pulmonary fibrosis in patients with H1N1 pneumonia: role of follow-up CT.

PURPOSE: Our aim was to evaluate the evolution of 20 patients with H1N1 pneumonia, focusing our attention on patients with severe clinical and radiological findings who developed post-acute respiratory distress syndrome (post-ARDS) pulmonary fibrosis. MATERIALS AND METHODS: Twenty adult patients (nine women and 11 men; mean age 43.5 ± 16.4 years) with a diagnosis of H1N1 infection confirmed by pharyngeal swab came to our attention from September to November 2009 and were followed up until September 2010. All patients were hospitalised in consideration of the severity of clinical findings, and all underwent chest X-ray. Twelve of them underwent at least one computed tomography (CT) scan of the chest. RESULTS: In 75% of cases (15/20), there was complete resolution of the clinical and radiological findings. Twenty-five percent of patients (5/20) developed acute respiratory distress syndrome (ARDS), which progressed to predominantly peripheral pulmonary fibrosis in 10% (2/20; one died and one had late-onset pulmonary fibrosis, documented on day 68). Moreover, in one patient with a CT diagnosis of pulmonary fibrosis, we observed progressive regression of radiological findings over 4 months of follow-up. CONCLUSIONS: In patients with H1N1 pneumonia, post-ARDS pulmonary fibrosis is not a rare complication. Therefore, a CT scan should be performed in all patients with severe clinical findings. Our study demonstrated that in these patients, fibrosis could present a different spatial distribution and a different temporal trend, with delayed late onset; moreover, in one case, the signs of interstitial lung disease partially regressed over time. Therefore, CT should be considered not only in the diagnostic stage, but also during the follow-up.

A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila.

CRISPR-based gene-drives have been proposed for managing insect populations, including disease-transmitting mosquitoes, due to their ability to bias their inheritance toward super-Mendelian rates (>50%). Current technologies use a Cas9 that introduces DNA double-strand breaks into the opposing wild-type allele to replace it with a copy of the gene-drive allele via DNA homology-directed repair. However, the use of different Cas9 versions is unexplored, and alternative approaches could increase the available toolkit for gene-drive designs. Here, we report a gene-drive that relies on Cas9 nickases that generate staggered paired nicks in DNA to propagate the engineered gene-drive cassette. We show that generating 5' overhangs in the system yields efficient allelic conversion. The nickase gene-drive arrangement produces large, stereotyped deletions that are advantageous to eliminate viable animals carrying small mutations when targeting essential genes. Our nickase approach should expand the repertoire for gene-drive arrangements aimed at applications in mosquitoes and beyond.