Highlights

Abstract

Graphical abstract

Keywords

1. Introduction

2. Literature search strategy & statistics

3. Historical framework: early hormetic findings

4. Radiation – UV and ionizing

5. Pollution and pollen

6. Polyamines

7. Discussion

8. Conclusion

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgement

Appendix A. Supplementary data

References

Abstract

This paper evaluated the occurrence of hormetic dose responses in pollen reported over the past eight decades. Hormetic doses responses were induced by a wide range of chemical and physical agents in 34 plant species for pollen germination and pollen tube growth/elongation. Agents inducing such hormetic dose/concentration responses in pollen included nutrients, growth-promoting agents, plant and animal hormones, toxic substances, including heavy metals such as cadmium, gaseous pollutants such as ozone, as well as ionizing and non-ionizing radiation. This paper provides further evidence for the broad generality of the hormesis dose response, supporting substantial prior findings that the hormetic response is independent of biological model, inducing agent, and endpoints measured. Given the widespread potential of inducing hormetic dose responses in pollen, these findings indicate the need to explore their emerging biological, ecological, agricultural, economic and public health implications.

1. INTRODUCTION

In flowering plants, the reproduction process is mediated by the pollen tube. This tubular structure provides the vehicle by which two immobile sperm cells are transported to the ovule, the location of two female reproduction cells (Sprunck, 2010; Dresselhaus and Franklin-Tong, 2013; Vogler et al., 2014). The pollen tube develops only after the pollen grain is transported, attaches, and then rehydrates on the receptive papillae that reside on the top of the stigma. After this process, the pollen grain becomes activated to form the so-called pollen tube, which may be characterized as a tubular protrusion that is chemically directed via complex physical and sensory processes through the pistil, eventually reaching the female gametophyte. At this point in the process, the male reproductive cells finally arrive at the target destination and initiate the process of fertilization (Palanivelu and Tsukamoto, 2012). Beyond their essential role in fertilization, pollen tubes are widely used to assess biological polarity, with broad applications to other cell types and biological processes. Of considerable theoretical and practical significance is that the pollen grain displays a self-organizing system that permits pollen to germinate, producing pollen tubes in vitro within an external signal mediated framework. The pollen germination rates, pollen tube growth rates, and final tube length are typically lower in in vitro studies than those observed in whole plants, most likely due to the absence of female factors that affect pollen germination, tube growth and growth directionality (Johnson and Presse, 2002; Chae and Lord, 2011). The selection of plants for pollen studies is affected by many species-specific processes as there are many factors that affect pollen tube growth, including the morphology of the reproductive tract (e.g. the stigma, style, the septum epidermis, and the funiculus). The nutrient containing extracellular matrix (ECM) contains a nutrient mixture that is important in enhancing pollen tube elongation Journal Pre-proof Journal Pre-proof 4 (Johnson and Presse, 2002; Chae and Lord, 2011). However, many studies employ bi-cellular pollen from species such as tobacco, which yields reliable findings (Volger et al., 2014).

Acknowledgments

Supplemental Information

References

Main Text

A novel coronavirus (CoV) named ‘‘2019 novel coronavirus’’ or ‘‘2019-nCoV’’ by the World Health Organization (WHO) is responsible for the recent pneumonia outbreak that started in early December, 2019 in Wuhan City, Hubei Province, China (Huang et al., 2020; Zhou et al., 2020; Zhu et al., 2020). This outbreak is associated with a large seafood and animal market, and investigations are ongoing to determine the origins of the infection. To date, thousands of human infections have been confirmed in China along with many exported cases across the globe (China CDC, 2020).

Coronaviruses mainly cause respiratory and gastrointestinal tract infections and are genetically classified into four major genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (Li, 2016). The former two genera primarily infect mammals, whereas the latter two predominantly infect birds (Tang et al., 2015). Six kinds of human CoVs have been previously identified. These include HCoV-NL63 and HCoV229E, which belong to the Alphacoronavirus genus; and HCoV-OC43, HCoVHKU1, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV), which belong to the Betacoronavirus genus (Tang et al., 2015). Coronaviruses did not attract worldwide attention until the 2003 SARS pandemic, followed by the 2012 MERS and, most recently, the 2019-nCoV outbreaks (China CDC, 2020; Song et al., 2019). SARS-CoV and MERS-CoV are considered highly pathogenic (Cui et al., 2019), and it is very likely that both SARS-CoV and MERS-CoV were transmitted from bats to palm civets (Guan et al., 2003) or dromedary camels (Drosten et al., 2014), and finally to humans (Cui et al., 2019).

The genome of coronaviruses, whose size ranges between approximately 26,000 and 32,000 bases, includes a variable number (from 6 to 11) of open reading frames (ORFs) (Song et al., 2019). The first ORF representing approximately 67% of the entire genome encodes 16 non-structural proteins (nsps), while the remaining ORFs encode accessory proteins and structural proteins (Cui et al., 2019). The four major structural proteins are the spike surface glycoprotein (S), small envelope protein (E), matrix protein (M), and nucleocapsid protein (N). The spike surface glycoprotein plays an essential role in binding to receptors on the host cell and determines host tropism (Li, 2016; Zhu et al., 2018). The spike proteins of SARS-CoV and MERS-CoV bind to different host receptors via different receptor-binding domains (RBDs). SARSCoV uses angiotensin-converting enzyme 2 (ACE2) as one of the main receptors (Ge et al., 2013) with CD209L as an alternative receptor (Jeffers et al., 2004), whereas MERS-CoV uses dipeptidyl peptidase 4 (DPP4, also known as CD26) as the primary receptor. Initial analysis suggested that 2019-nCoV has a close evolutionary association with the SARSlike bat coronaviruses (Zhou et al., 2020). Here, based on the first three determined genomes of the novel coronavirus (2019-nCoV), namely Wuhan/ IVDC-HB-01/2019 (GISAID accession ID: EPI_ISL_402119) (HB01), Wuhan/IVDCHB-04/2019 (EPI_ISL_402120) (HB04), and Wuhan/IVDC-HB-05/2019 (EPI_ ISL_402121) (HB05), an in-depth genome annotation of this virus was performed with a comparison to related coronaviruses, including 1,008 human SARSCoV, 338 bat SARS-like CoV, and 3,131 human MERS-CoV, whose genomes were published before January 12, 2020 (release date: September 12, 2019) from Virus Pathogen Database and Analysis Resource (ViPR) (http://www.viprbrc. org/) and NCBI.

Declaration of Competing Interest

References

Background: A novel coronavirus (2019-nCoV) associated with human to human transmission and severe human infection has been recently reported from the city of Wuhan in China. Our objectives were to characterize the genetic relationships of the 2019-nCoV and to search for putative recombination within the subgenus of sarbecovirus.

Methods: Putative recombination was investigated by RDP4 and Simplot v3.5.1 and discordant phylogenetic clustering in individual genomic fragments was confirmed by phylogenetic analysis using maximum likelihood and Bayesian methods.

Results: Our analysis suggests that the 2019-nCoV although closely related to BatCoV RaTG13 sequence throughout the genome (sequence similarity 96.3%), shows discordant clustering with the Bat_SARS-like coronavirus sequences. Specifically, in the 5′-part spanning the first 11,498 nucleotides and the last 3′-part spanning 24,341–30,696 positions, 2019-nCoV and RaTG13 formed a single cluster with Bat_SARS-like coronavirus sequences, whereas in the middle region spanning the 3′-end of ORF1a, the ORF1b and almost half of the spike regions, 2019-nCoV and RaTG13 grouped in a separate distant lineage within the sarbecovirus branch.

Conclusions: The levels of genetic similarity between the 2019-nCoV and RaTG13 suggest that the latter does not provide the exact variant that caused the outbreak in humans, but the hypothesis that 2019-nCoV has originated from bats is very likely. We show evidence that the novel coronavirus (2019-nCov) is not-mosaic consisting in almost half of its genome of a distinct lineage within the betacoronavirus. These genomic features and their potential association with virus characteristics and virulence in humans need further attention.

1- Introduction

2- Material and methods

3- Results

4- Discussion

References

Abstract

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites.

Introduction

Mites and ticks are members of the chelicerates, the largest group of terrestrial animals after insects. The two-spotted spider mite, T. urticae, and other spider mites are important crop pests worldwide. This herbivore species is at the extreme end of the generalist-to specialist spectrum and can feed on a staggering 1,100 plant species. Not surprisingly, it is currently reported as the ‘most resistant’ pest worldwide, as it developed resistance to more than 90 acaricides (Mota-Sanchez and Wise, 2019; Van Leeuwen and Dermauw, 2016; Van Leeuwen et al., 2015). In 2011, a 90 Mb high-quality Sanger-sequenced genome became available for this species (Grbic et al., 2011). This allowed to disentangle some of the molecular mechanisms underlying resistance, whether to man-made pesticides or plant secondary compounds. The extreme adaptation potential of T. urticae was associated with specific gene expansions in known detoxification enzyme families, such as cytochrome P450 monooxygenases, glutathione-S-transferases, carboxyl-choline esterases, an unexpected repertoire of ABC and MFS transporters, and a proliferation of cysteine peptidases (Dermauw et al., 2013a; Dermauw et al., 2013b; Grbic et al., 2011; Santamaría et al., 2012).

1- A brief history of CRISPR

2- CRISPR/Cas9: biology, functionality, and applications

3- Therapeutic targets for ex vivo cell-based CRISPR/Cas9 genome editing

4- CRISPR therapy in clinical trials

5- Challenges and opportunities

References

Abstract

The recently developed CRISPR/Cas9 technology has revolutionized the genome engineering field. Since 2016, increasing number of studies regarding CRISPR therapeutics have entered clinical trials, most of which are focusing on the ex vivo genome editing. In this review, we highlight the ex vivo cell-based CRISPR/Cas9 genome editing for therapeutic applications. In these studies, CRISPR/Cas9 tools were used to edit cells in vitro and the successfully edited cells were considered as therapeutics, which can be introduced into patients to treat diseases. Considering a large number of previous reviews have been focused on the CRISPR/Cas9 delivery methods and materials, this review provides a different perspective, by mainly introducing the targeted conditions and design strategies for ex vivo CRISPR/Cas9 therapeutics. Brief descriptions of the history, functionality, and applications of CRISPR/Cas9 systems will be introduced first, followed by the design strategies and most significant results from previous research that used ex vivo CRISPR/Cas9 genome editing for the treatment of conditions or diseases. The last part of this review includes general information about the status of CRISPR/Cas9 therapeutics in clinical trials. We also discuss some of the challenges as well as the opportunities in this research area.

A brief history of CRISPR

The term CRISPR, or the clustered regularly interspaced palindromic repeats, was first used by Jansen et al., in 2002 to describe a novel family of repetitive DNA sequences presented in the genomes of prokaryotes [1]. These unusual repeated sequences were first detected in 1987 by Nakata et al. in Escherichia coli [2], and then recognized to be widespread in archaea and bacteria by Mojica et al., in 2000 [3]. After Jansen et al. identified the CRISPR-associated (Cas) genes in 2002 [1], Mojica et al. [4], Vergnaud et al. [5], and Bolotin et al. [6] revealed the exrtachromosomal and phage-associated origins of the spacers that separate the individual direct repeats, in 2005. Speculations about CRISPR arrays as immune memory and defense mechanism against virus envisions were then proposed [6,7], while the first experimental evidence for CRISPR/Cas system based adaptive immunity was reported by Horvath et al., in 2007 [8]. Following the series discoveries on the basic function and mechanism of CRISPR systems [9–12], in 2010 Moineau et al. demonstrated the Cas9 enzyme (formerly named Cas5, COG3513, Csn1 or Csx12), which is guided by spacer sequences, cleaves target DNA [13]. In 2012, Doudna, Charpentier et al. [14], and Siksnys et al. [15] proved that Streptococcus pyogenes and Streptococcus thermophiles Cas9 can be guided by CRSIPR RNAs (crRNAs) to cleave target DNA in vitro, through forming a double-strand break (DSB). In 2013, Zhang et al. [16], Church et al. [17], and Doudna et al. [18] independently demonstrated the applications of engineered CRISPR/Cas9 systems for genome editing in mammalian cells. This represented a giant leap forward in the genome engineering field and triggered a tremendous number of studies regarding the use of the CRISPR/Cas9 platform for eukaryotic gene editing in a wide range of species, including flies [19,20], zebrafish [21–24], frogs [25], mice [26–28], rats [26,29], pigs [30], and monkeys [31,32].

1- Introduction

2- Multi-layer NPs for CRISPR-Cas9 delivery

3- Novel building blocks for multi-layer NPs

4- Conclusions and perspective

References

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) has great potential to revolutionize biomedical research and disease therapy. The specific and efficient genome editing strongly depends on high efficiency of delivery of the CRISPR payloads. However, optimization of CRISPR delivery vehicles still remains a major obstacle. Recently, various non-viral vectors have been utilized to deliver CRISPR tools. Many of these vectors have multi-layer structures assembled. In this review, we will introduce the development of CRISPR-Cas9 systems and their general therapeutic applications by summarizing current CRISPR-Cas9 based clinical trials. We will highlight the multi-layer nanoparticles (NPs) that have been developed to deliver CRISPR cargos in vitro and in vivo for various purposes, as well the potential building blocks of multi-layer NPs. We will also discuss the challenges in making the CRISPR tools into viable pharmaceutical products and provide potential solutions on efficiency and biosafety issues.

Introduction

In recent years, a series of programmable DNA nuclease-dependent genome editing techniques have been developed to enable efficient genetic manipulation in eukaryotes, particularly in mammalian cells [1-3]. These genome-editing techniques used for gene knock-out or knock-in predominately include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system [4-7]. These technologies all employ endonucleases to induce double strand breaks (DSB) at intended sites in the genome, followed by DSB repair through different mechanisms including non-homologous end joining (NHEJ), homologous recombination (HR), homology-mediated end joining (HMEJ) and microhomology-mediated end joining (MMEJ) [8]. Among these genome editing techniques, CRISPR-Cas9 is a gene editing system derived from the bacterial adaptive immune system, which is capable of combating the invasive viral genome under the guidance of short sequence RNA. CRISPR-Cas9 system is highly versatile because of its simplicity and high efficiency, moreover, any genome sequence theoretically can be edited by this system. In principle, the whole genome can be site-specifically modified at the genetic level in cells and tissues with CRISPR-Cas9 system, which further allows researchers to study the relationship between gene mutations and biological phenotypes. CRISPR-Cas9 system has driven innovative applications from basic biology research to biotechnology and medicine [9].

1- Introduction

2- Genome repair mechanisms

3- Intracellular free exogenous reporter system

4- Cell genome integrated endogenous reporter system

5- Outlooks

References

Abstract

Genome editing has become a vital tool in medical biology research. The critical mission of facilitating the progress of genome editing is to enrich different genes into positive edited cells quickly and effectively and carry out the targeted research. In recent years, researchers have established various reporter systems for selection and enrichment of editing-induced positive cells, which are based on genome repair mechanisms, such as non-homologous end joining, homology-directed repair, single strand annealing and inversion, and the principle of the expression of fluorescent protein or resistance tag after genome repair. The T7E1 assay or sequencing method can analyze the mutation of enriched cells with the results of lower background signals and higher mutation ratio. Therefore, these reporter systems can profit the characterization of genome editing effectiveness. Besides, positive cells can be cultured continuously, so this technology possesses a promising prospect in mutated cell line construction and the research of mutated cell functions. This article summarized the design principles and applications of these reporter systems and would provide a reference to construct a more perfecte evaluating system for genome editing.

Introduction

Genetic engineering started when monkey virus SV40 and E. coli bacteriophage λDNA spliced into a loop for the first time[1]. Subsequently, the discovery and research of genome repair mechanisms such as homologous recombination (HR), non-homologous end joining (NHEJ), and endonucleases provided a theoretical basis for modern genome editing technology[2,3]. In recent years, researchers have found some nucleases based on long sequence recognition, such as meganuclease[4‒7] or homing endonuclease, zinc finger nucleases (ZFNs)[8‒12], transcription activator-like effector nucleases (TALENs)[13‒17], structure-guided nucleases (SGN)[18] and clustered regularly interspaced short palindromic repeats associated protein Cas9 (CRISPR/Cas9)[19‒22]. These nucleases can accurately recognize the target sequence and induce double-strand break (DSB), and then through homologydirected repair (HDR)[23] or NHEJ[24] and other mechanisms to edit the target sequence. Genome editing technology has been applied in various fields, such as the establishment of disease models[26‒28], gene therapy[29], and gene screening[30] through different transfection techniques, such as liposomes, electroporation, viral packaging, and transmembrane peptides[25]. However, the off-target effects inherent in genome editing tools[31,32] may induce side effects such as non-target editing, erroneous phenotypes, or lethality in practical applications.

1- Introduction

2- Conventional approach to generate genetically engineered livestock

3- Development of genome editing technology

4- Application of genome editing technology in livestock

5- Genetically engineered livestock produced using genome editing technology

6- Future prospective and current limitations

7- Conclusion

References

Abstract

Historically, genetic engineering in livestock proved to be challenging. Without stable embryonic stem cell lines to utilize, somatic cell nuclear transfer (SCNT) had to be employed to produce many of the genetically engineered (GE) livestock models. Through the genetic engineering of somatic cells followed by SCNT, GE livestock models could be generated carrying site-specific modifications. Although successful, only a few GE livestock models were generated because of low efficiency and associated birth defects. Recently, there have been major strides in the development of genome editing tools: Zinc-Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENS), and Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (Cas9) system. These tools rely on the generation of a double strand DNA break, followed by one of two repair pathways: non-homologous end joining (NHEJ) or homology directed repair (HDR). Compared to the traditional approaches, these tools dramatically reduce time and effort needed to establish a GE animal. Another benefit of utilizing genome editing tools is the application of direct injection into developing embryos to induce targeted mutations, therefore, eliminating side effects associated with SCNT. Emerging technological advancements of genome editing systems have dramatically improved efficiency to generate GE livestock models for both biomedical and agricultural purposes. Although the efficiency of genome editing tools has revolutionized GE livestock production, improvements for safe and consistent application are desired. This review will provide an overview of genome editing techniques, as well as examples of GE livestock models for agricultural and biomedical purposes.

Introduction

54 The ability to engineer the genome has been limited in livestock species, in part due to 55 the lack of stable embryonic stem (ES) cells that can contribute to the germline. In the mouse, 56 targeted disruption of genes can be performed in ES cells, then the ES cells carrying desired 57 genotypes can be introduced into recipient blastocysts to generate chimeric mice that can 58 transmit the desired genotype to the germline [1-3]. This strategy has been effective, thus 59 establishing the mouse as the main model in biomedicine to elucidate the function of target 60 genes. Since true ES cells that can contribute to the germline have not been fully identified in 61 livestock species, the development of somatic cell nuclear transfer (SCNT) technology [4] 62 allowed researchers to generate genetically engineered (GE) livestock. Genetic modification of 63 somatic cells followed by SCNT allowed for the generation of GE livestock with targeted 64 modifications [5]. Although possible, efficiency to produce GE animals through the route is poor; 65 therefore, only selective GE models have been produced [6, 7].

1- Introduction

2- Common lethal cancers

3- Investigating miRNA profiling in cancers

4- MiR candidates for early phase group detection

5- Conclusion

References

Abstract

Cancer is considered as a challenging lethal agent around the world and its detection at early stages would help prevention of the high mortality. Among the widely used biomarkers in clinical diagnosis of cancer, extracellular non-coding RNAs as ribonucleic acid biomarkers serve as state-of-the-art candidates for molecular diagnosis. In that regard, microRNAs are of great priority mainly because of high variety and stability in body fluids. Accordingly, common miRNAs among most prevalent cancers could help us (pre)diagnose cancer with high accuracy in target samples. In this study, common lethal cancers to humans were investigated in case of miRNA profiles to determine the possible common correlation between miRNA up-regulation or down-regulation (as a ribonucleic acid biomarker) and developing the cancers. It was shown that among the investigated miRNAs, five typical extracellular miRNAs (miR-18a, miR-21, miR-155, miR-221, and miR-375) dysregulation are predominant in most cancer varieties comprising breast, colon, lung, prostate, pancreas, gastric, ovarian, esophagus and liver. This could serve as an appropriate target site for developing point-of-care approaches for cancer detection e.g. designing diagnostic biosensor-based microarrays or kits for both quantification and qualification of the biomarkers. Besides, the miRNA candidates could be efficiently applied to cancer therapeutic approaches.

Introduction

Cancer keeps on being a remarkable cause of worldwide mortality in spite of many years of effort and cost. Deadliest types of cancers to human includes pancreas, liver, lung and bronchus, prostate/breast, colon, rectum and ovary [1–3]. Most of cancers do not induce clinical symptoms until in the later stages when the therapeutic treatment is no longer an option. However, detection of cancer at first stages, regardless of its origin, greatly increases the chance of effective treatments. In spite of much research in cancer biomarkers, only a few are considered for early diagnosis of common cancers. In addition, there is not a general biomarker for population screening in order to detect both asymptomatic cancers and early-staged cancers. Hence, investigating more sensitive and non-invasive biomarkers is still a promising challenge in cancer diagnosis and prognosis [4]. Accessing circulating cancer biomarkers through biological fluids using liquid biopsy seems to propose a promising cost-effective and non-invasive solution. Literally, cf-miRNAs are considered as ideal circulating cancer biomarkers due to their ease of access and quantification, besides integration with other macromolecules e.g. proteins, and high stability in plasma [5, 6]. MiRNAs are endogenous, small (18–24 nt), non-coding (nc) RNA molecules that play important regulatory roles in cell proliferation, apoptosis, metastasis and angiogenesis [7]. Dysregulated miRNA(s) have both oncogenic and tumor-suppressing effects depending on their corresponding targets. The increase or decrease of miRNAs is related to the role of their target genes in cancer progression or regression. MiRNAs are mainly categorized in two groups: 1) OncomiRs that are up-regulated in cancers to target protein-coding transcripts in tumor suppressive pathways, and consequently increase cell proliferation, invasion and metastasis. 2) Tumor-suppressor miRNAs that repress the oncogenic genes and their down-regulation in cancer increases the oncogenic activity [8]. MiRNAs show various expression patterns in tissue and blood of many cancers in comparison with normal samples. Furthermore, miRNA profiles in cancers with various tissue origins are distinct from each other [9].

1- Introduction

2- Potential target diseases of genome editing in the cardiovascular field

3- Molecular mechanisms and strategies of genome editing to treat or prevent diseases

4- Current hurdles for therapeutic genome editing

5- Conclusions

References

Abstract

During the past decade, developments in genome editing technology have fundamentally transformed biomedical research. In particular, the CRISPR/Cas9 system has been extensively applied because of its simplicity and ability to alter genomic sequences within living organisms, and an ever increasing number of CRISPR/Cas9-based molecular tools are being developed for a wide variety of applications. While genome editing tools have been used for many aspects of biological research, they also have enormous potential to be used for genome editing therapy to treat a broad range of diseases. For some hematopoietic diseases, clinical trials of therapeutic genome editing with CRISPR/Cas9 are already starting phase I. In the cardiovascular field, genome editing tools have been utilized to understand the mechanisms of diseases such as cardiomyopathy, arrythmia, and lipid metabolism, which now open the door to therapeutic genome editing. Currently, therapeutic genome editing in the cardiovascular field is centered on liver-targeting strategies to reduce cardiovascular risks. Targeting the heart is more challenging. In this review, we discuss the potential applications, recent advances, and current limitations of therapeutic genome editing in the cardiovascular field.

Introduction

The development of genome editing technology was one of the biggest advances in biology in the last decade, and it has revolutionized biomedical research [1-4]. Compared to methods using conventional homologous recombination, which also can modify genomic sequences and are commonly used to generate genetic models, the new genome editing tools have made it much easier and faster to alter genomic DNA sequences within the genome of living organisms. Basically, the genome editing tools harness the endogenous repair process of DNA double-strand breaks (DSBs) generated by programmable nucleases such as zinc finger nucleases (ZFNs) [5], transcription activator-like effector nucleases (TALENs) [6, 7], and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) [2-4] nucleases. In particular, the key initial technological breakthrough was the application of CRISPR/Cas9 system, which was originally discovered as a part of the immune system within bacteria, to eukaryotic genome editing [8]. Because of their simplicity and efficiency, CRISPR/Cas9 genome editing tools have been widely adopted in various scientific fields.

1- Introduction

2- Methodology

3- Results

4- Discussion

5- Conclusion

References

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables precision editing of the genome of many plant species. Developing robust gene editing tools in banana and plantain would pave the way for the improvement of these crops. Here, we developed efficient CRISPR/Cas9 genome editing protocol for banana and plantain using multiple gRNAs targeting phytoene desaturase (PDS) gene. CRISPR/Cas9 construct containing two gRNAs was delivered into embryogenic cell suspension cultures of banana cultivar ‘Sukali Ndiizi’ and plantain cultivar ‘Gonja Manjaya’ by Agrobacterium-mediated transformation. The regenerated genome-edited events of ‘Sukali Ndiizi’ and ‘Gonja Manjaya’ showed albino and variegated phenotypes indicating mutations at the targeted sites disrupting the function of PDS gene. The majority of events (52/77 for ‘Sukali Ndiizi’ and 16/17 for ‘Gonja Manjaya’) were albino. Sequencing of the target sites confirmed presence of indels in all the 18 events sequenced demonstrating mutation efficiency of 100 % in both cultivars. The majority of events (6/8) of ‘Gonja Manjaya’ showed indels at both the target sites of PDS gene, however only 2/10 events of ‘Sukali Ndiizi’ showed indels at both sites, with one event (S24) having a knockout of large fragment (723 bp) indicating that both gRNAs were effective. Several of the albino events of both ‘Sukali Ndiizi’ and ‘Gonja Manjaya’ showed homozygous mutations. Further sequencing of four potential off-target sites in five events showed no mutations indicating CRISPR/Cas9 based editing in banana and plantain is targeted and precise with a very low probability of off-target sites. This study could provide a methodological framework for single or multiple knockouts in banana and plantain.

Introduction

Genome editing technologies using specific nucleases have been developed as effective genetic engineering methods for targeted mutations at specific sites in the genome of an organism [1]. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated protein 9) based genome editing is the emerging most powerful tool with ability to create targeted mutations in the genome of crops and to understand the functional biology. These nucleases respond in a target-specific manner and induce a double-strand break (DSB) in the gene sequence [2]. The DSB is repaired either by homologous recombination (HR) or errorprone non-homologous end-joining (NHEJ) mechanisms, which may lead to disruption of gene function [3]. CRISPR/Cas9 system has emerged as a potent genome editing tool due to its simplicity, design flexibility, and high efficiency, and has been applied successfully in several plant species [4]. It is based on the type II CRISPR/Cas immune system of bacteria that protects against invading DNA viruses and/or plasmids. The CRISPR/Cas9 molecular immunity system is made up of the Cas9 endonuclease from Streptococcus pyogenes and a single guide RNA (sgRNA). The sgRNA directs the Cas9 endonuclease to a target sequence complementary to the 20 nucleotides preceding the protospacer-adjacent motif (PAM) NGG or NAG, which is required for Cas9 activity [5-6]. Multiplexing of CRISPR/Cas9 system by integrating two or more gRNAs, which are distance apart, is useful in generating large deletions in plants [7].

Introduction

Genomic and structure

Epidemiology

Clinical characteristics

Treatments for 2019-nCoV

Discussion

Acknowledgement

References

Abstract

A novel coronavirus designated as 2019-nCoV hit the central Chinese city of Wuhan in late December 2019, and subsequently spread rapidly to all provinces of China and multiple countries. Up to 0:00 am February 9, 2020, a total of 37,287 cases have been confirmed infection of 2019-nCoV in China mainland, and 302 cases have also been cumulatively reported from 24 countries. According to the latest data, a total of 813 deaths occurred in China mainland, with the mortality reaching approximately 2.2%. At present, there is no vaccine or specific drugs for human coronavirus, so that it’s critical to understand the nature of the virus and its clinical characteristics to response to the 2019-nCoV outbreak. Thus, we summarize the not much but timely reports on the 2019-nCoV in the present study, briefly but comprehensively.

Introduction

Coronaviruses (CoVs) are enveloped, single positive stranded RNA virus, which belong to the subfamily Coronavirinae. The CoVs genome, ranging from 26 to 32 kilobases in length, is probably the largest viral RNA known (1,2). Previously, there are six CoVs known to cause human diseases and can be divided into low pathogenic and highly pathogenic CoVs (2,3). The low pathogenic CoVs, including 229E, HKU1, OC43 and NL63, account for 10% to 30% of upper respiratory tract infections and typically cause mild respiratory diseases (3,4). In contrast, the highly pathogenic CoVs, including Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) CoV, predominantly infect lower airways and cause fatal pneumonia (2,5).

SARS-CoV emerged as a new human infection in South China in November 2002 and ended in July 2003. It infected 8,096 people and caused 774 deaths with an overall mortality rate of about 9.6% (6,7). MERS-CoV, another highly pathogenic CoV, first emerged in Saudi Arabiawas, has caused a total of 2494 laboratory-confirmed cases and 858 deaths from 27 countries (mortality rate, 34.4%) since September 2012. (http://www.who.int/emergencies/mers-cov/en/) (8). These two highly pathogenic β-CoVs have posed a substantial threat to public health.