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Gene editing staves off deafness in mice.
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Applications and challenges of CRISPR-Cas gene-editing to disease treatment in clinics.
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) are efficient tools for targeting specific genes for laboratory research, agricultural engineering, biotechnology, and human disease treatment. Cas9, by far the most extensively used gene-editing nuclease, has shown great promise for the treatment of hereditary diseases, viral infection, cancers, and so on. Recent reports have revealed that some other types of CRISPR-Cas systems may also have surprising potential to join the fray as gene-editing tools for various applications. Despite the rapid progress in basic research and clinical tests, some underlying problems present continuous, significant challenges, such as editing efficiency, relative difficulty in delivery, off-target effects, immunogenicity, etc. This article summarizes the applications of CRISPR-Cas from bench to bedside and highlights the current obstacles that may limit the usage of CRISPR-Cas systems as gene-editing toolkits in precision medicine and offer some viewpoints that may help to tackle these challenges and facilitate technical development. CRISPR-Cas systems, as a powerful gene-editing approach, will offer great hopes in clinical treatments for many individuals with currently incurable diseases.
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A Need for Better Understanding Is the Major Determinant for Public Perceptions of Human Gene Editing.
The CRISPR/Cas system could provide an efficient and reliable means of editing the human genome and has the potential to revolutionize modern medicine; however, rapid developments are raising complex ethical issues. There has been significant scientific debate regarding the acceptability of some applications of CRISPR/Cas, with leaders in the field highlighting the need for the lay public's views to shape expert discussion. As such, we sought to determine the factors that influence public opinion on gene editing. We created a 17-item online survey translated into 11 languages and advertised worldwide. Topic modeling was used to analyze textual responses to determine what factors influenced respondents' opinions toward human somatic or embryonic gene editing, and how this varied among respondents with differing attitudes and demographic backgrounds. A total of 3,988 free-text responses were analyzed. Respondents had a mean age of 32 (range, 11-90) years, and 37% were female. The most prevalent topics cited were Future Generations, Research, Human Editing, Children, and Health. Respondents who disagreed with gene editing for health-related purposes were more likely to cite the topic Better Understanding than those who agreed to both somatic and embryonic gene editing. Respondents from Western backgrounds more frequently discussed Future Generations, compared with participants from Eastern countries. Religious respondents did not cite the topic Religious Beliefs more frequently than did nonreligious respondents, whereas Christian respondents were more likely to cite the topic Future Generations. Our results suggest that public resistance to human somatic or embryonic gene editing does not stem from an inherent mistrust of genome modification, but rather a desire for greater understanding. Furthermore, we demonstrate that factors influencing public opinion vary greatly amongst demographic groups. It is crucial that the determinants of public attitudes toward CRISPR/Cas be well understood so that the technology does not suffer the negative public sentiment seen with previous genetic biotechnologies.
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The Potential Therapeutic Applications of CRISPR/Cas9 in the Treatment of Gastrointestinal Cancers.
Gastrointestinal (GI) cancer is one the most prevalent types of cancer. Despite current chemotherapy's success, patients with GI cancer continue to have a dismal outcome. The onset and progression of cancer are caused by alterations and the abnormal expression of several families of genes, like tumor-suppressor genes, oncogenes, and chemotherapy-resistant genes. The final purpose of tumor therapy is to inhibit cellular development by modifying mutations and editing the irregular expression of genes It has been reported that CDH1, TP53, KRAS, ARID1A, PTEN, and HLA-B are the commonly mutated genes in GI cancer. Gene editing has become one potential approach for cases with advanced or recurrent CRC, who are nonresponsive to conventional treatments and a variety of driver mutations along with progression cause GI progression. CRISPR/Cas9 technique is a reliable tool to edit the genome and understand the functions of mutations driving GI cancer development. CRISPR/Cas9 can be applied to genome therapy for GI cancers, particularly with reference to molecular-targeted medicines and suppressors. Moreover, it can be used as a therapeutic approach by knocking in/out multiple genes. The use of CRISPR/ Cas9 gene editing method for GI cancer therapy has therefore resulted in some improvements. There are several research works on the role of CRISPR/Cas9 in cancer treatment that are summarized in the following separate sections. Here, the use of CRISPR/Cas9-based genome editing in GI and the use of CRISPR/Cas9 is discussed in terms of Targeting Chemotherapy Resistance-related Genes like; KRAS, TP53, PTEN, and ARID1A.
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The CRISPR-baby scandal: what's next for human gene-editing.
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CRISPR-Cas9 mediated knockout of the white gene in the bluetongue virus vector, Culicoides sonorensis (biting midge).
Culicoides biting midges are small blood feeding insects responsible for the transmission of important arthropod-borne viruses (arboviruses) such as bluetongue virus (BTV), Schmallenberg virus (SBV) and epizootic hemorrhagic disease virus (EHDV), which cause major losses to livestock production worldwide. Culicoides sonorensis is the primary vector of BTV in North America and one of the few Culicoides species to be colonised and reared in artificial conditions. Gene editing technology has been used to explore virus-vector interactions in other vector groups, particularly within mosquitoes. Despite the availability of a reference genome since 2018, to date there have been no reports of gene editing in C. sonorensis. Here, we report the first instance of gene editing in C. sonorensis, achieved by intrathoracic injection of adult females with Cas9 and sgRNAs targeting the white gene. We generated heritable mutations in the white gene which produced both white eye and red eye phenotypes and went on to establish a homozygous knockout line carrying a single mutation. We observed gene editing efficiencies of up to 12.3%, making this an efficient protocol for genetic manipulation of Culicoides biting midges, opening the door to functional genomics studies and the development of control strategies in these important and understudied disease vectors.
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Prime Editing for Precise Genome Engineering in Drosophila.
Editing the Drosophila genome is incredibly useful for gene functional analysis. However, compared to gene knockouts, precise gene editing is difficult to achieve. Prime editing, a recently described CRISPR/Cas9-based technique, has the potential to make precise editing simpler and faster, and produce less errors than traditional methods. Initially described in mammalian cells, prime editing is functional in Drosophila somatic and germ cells. Here, we outline steps to design, generate, and express prime editing components in transgenic flies. Furthermore, we highlight a crossing scheme to produce edited fly stocks in less than 3 months.
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Breeding herbicide-resistant rice using CRISPR-Cas gene editing and other technologies.
The emergence of herbicide-resistant weeds in crop fields and the extensive use of herbicides have led to a decrease in rice (Oryza sativa) yields and an increase in production costs. To address these challenges, researchers have focused on the discovery of new germplasm resources with herbicide resistance. The most promising candidate genes have been functionally studied and applied in rice breeding. Here, we review recent progress in the breeding of herbicide-resistant rice. We provide examples of various techniques used to breed herbicide-resistant rice, such as physical and chemical mutagenesis, genetic transformation, and CRISPR-Cas-mediated gene editing. We highlight factors involved in the breeding of herbicide-resistant rice, including target genes, rice varieties, degrees of herbicide resistance, and research tools. Finally, we suggest methods for breeding herbicide-resistant rice that could potentially be used for weed management in direct-seeding farm systems.
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Strong and tunable anti-CRISPR/Cas activities in plants.
CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti-CRISPR proteins as tools to prevent CRISPR/Cas-mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti-CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a-mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas-based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants.
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Gene editing to improve legume-rhizobia symbiosis in a changing climate.
In the last three years, several gene editing techniques have been developed for both model and crop legumes. CRISPR-Cas9-based tools, in particular, are outpacing other comparable gene editing technologies used in legume hosts and their microbial symbionts to understand the molecular basis of symbiotic nitrogen-fixation. Gene editing has helped identify new gene functions, validate genetic screens, resolve gene redundancy, examine the role of tandemly duplicated genes, and investigate symbiotic signaling networks in non-model plants. In this review, we discuss the advances made in understanding the legume-rhizobia symbiosis through the use of gene editing and highlight studies conducted under varying environmental conditions. We reason that future climate-hardy legumes must be able to better integrate environmental signals with nitrogen fixation by fine-tuning long distance signaling, continuing to select efficient rhizobial partners, and adjusting their molecular circuitry to function optimally under variable light and nutrient availability and rising atmospheric carbon dioxide.
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