Браузер не даёт сохранять данные (например, приватный режим): закладки пропадут после закрытия вкладки.
Поиск статей PubMed
Ключевые слова, автор, журнал, годы — результаты из PubMed, аннотации с переводом на русский, закладки в браузере.
Найдено: 26 219
Страница 734 из 1000-
Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba.
Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype's characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.
-
Therapeutic application of sequence-specific binding molecules for novel genome editing tools.
Genome editing has been expected to widely increase the available treatment options for various diseases and permit pharmaceutical interventions in previously untreatable conditions. The availability of genome editing tools was dramatically increased by the development of the CRISPR-Cas9 system. However, a number of issues limit the use of the CRISPR-Cas9 system and other gene-editing tools in the clinical treatment of diseases. This review summarized the history and types of genome editing tools and limitations of their use. In addition, the study addressed several next-generation technologies aiming to overcome the limitations of current gene therapy protocols in an effort to accelerate the clinical development of potential treatment options. This review has provided an extensive foundation of the current state of genome editing technology and its clinical development. This review also indicate that the study additionally highlighted the need for multidisciplinary approaches to overcome current bottlenecks in the development of genome editing.
-
Challenges and Advances in Genome Editing Technologies in Streptomyces.
The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.
-
A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells.
Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3-6 weeks in order to understand genetic determinants of disease and precision medicine.
-
Milestones of CRISPR/Cas9 system, promises and roadblocks.
In this issue of Cancer Treatment and Research Communications, Sarkar et al. describe CRISPR/Cas9-based gene-editing from its discovery to current views. Originally a bacterial adaptive immune system, CRISPR/Cas9 was adapted to mammalian cells as a tool to perform a wide range of actions given its capability of accurately targeting specific DNA loci. While the CRISPR/Cas9 system is readily used in laboratories as a research tool for a few years now, the first clinical trials have recently started utilizing this system in translational medicine. Although several obstacles have been resolved related to the usage of CRISPR/Cas9 on mammalian cells, the scientific community is facing new challenges with this technology.
-
Designing babies through gene editing: science or science fiction?
-
On recent advances in human engineering Provocative trends in embryology, genetics, and regenerative medicine.
Advances in embryology, genetics, and regenerative medicine regularly attract attention from scientists, scholars, journalists, and policymakers, yet implications of these advances may be broader than commonly supposed. Laboratories culturing human embryos, editing human genes, and creating human-animal chimeras have been working along lines that are now becoming intertwined. Embryogenic methods are weaving traditional in vivo and in vitro distinctions into a new "in vivitro" (in life in glass) fabric. These and other methods known to be in use or thought to be in development promise soon to bring society to startling choices and discomfiting predicaments, all in a global effort to supply reliably rejuvenating stem cells, to grow immunologically non-provocative replacement organs, and to prevent, treat, cure, or even someday eradicate diseases having genetic or epigenetic mechanisms. With humanity's human-engineering era now begun, procedural prohibitions, funding restrictions, institutional controls, and transparency rules are proving ineffective, and business incentives are migrating into the most basic life-sciences inquiries, wherein lie huge biomedical potentials and bioethical risks. Rights, health, and heritage are coming into play with bioethical presumptions and formal protections urgently needing reassessment.
-
CRISPR/Cas mediated genome editing in potato: Past achievements and future directions.
Genome engineering has been re-shaping plant biotechnology and agriculture. Crop improvement using the recently developed gene editing techniques is now easier, faster, and more precise than ever. Although considered to be a global food security crop, potato has not benefitted enough from diverse collection of these techniques. Unique genetic features of cultivated potatoes such as tetrasomic inheritance, high genomic heterozygosity, and inbreeding depression hamper conventional breeding of this important crop. Therefore, genome editing provides an excellent arsenal of tools for trait improvement in potato. Moreover, using specific transformation protocols, it is possible to engineer transgene free commercial varieties. In this review we first describe the past achievements in potato genome editing and highlight some of the missing aspects of these efforts. Then, we discuss about technical challenges of genome editing in potato and present approaches to overcome these difficulties. Finally, we talk about genome editing applications that have not been explored in potato and point out some of the missing venues in literature.
-
A Guanidinobenzol-Rich Polymer Overcoming Cascade Delivery Barriers for CRISPR-Cas9 Genome Editing.
The efficient cytosolic delivery of the CRISPR-Cas9 machinery remains a challenge for genome editing. Herein, we performed ligand screening and identified a guanidinobenzol-rich polymer to overcome the cascade delivery barriers of CRISPR-Cas9 ribonucleoproteins (RNPs) for genome editing. RNPs were stably loaded into the polymeric nanoparticles (PGBA NPs) by their inherent affinity. The polymer facilitated rapid endosomal escape of RNPs via a dynamic multiple-step cascade process. Importantly, the incorporation of fluorescence in the polymer helps to identify the correlation between cellular uptake and editing efficiency, increasing the efficiency up to 70% from the initial 30% for the enrichment of edited cells. The PGBA NPs efficiently deliver RNPs for in vivo gene editing via both local and systemic injections and dramatically reduce PCSK9 level. These results indicate that PGBA NPs enable the cascade delivery of RNPs for genome editing, showing great promise in broadening the therapeutic potential of the CRISPR-Cas9 technique.
-
Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha.
BACKGROUND: Ogataea polymorpha, a non-conventional methylotrophic yeast, has demonstrated significant potential for heterologous protein expression and the production of high-value chemicals and biopharmaceuticals. However, the lack of precise and efficient genome editing tools severely hinders the construction of cell factories. Although the CARISP-Cas9 system has been established in Ogataea polymorpha, the gene editing efficiency, especially for multiple genes edition, needs to be further improved. RESULTS: In this study, we developed an efficient CRISPR-Cpf1-mediated genome editing system in O. polymorpha that exhibited high editing efficiency for single gene (98.1 ± 1.7%), duplex genes (93.9 ± 2.4%), and triplex genes (94.0 ± 6.0%). Additionally, by knocking out non-homologous end joining (NHEJ) related genes, homologous recombination (HR) efficiency was increased from less than 30% to 90 ~ 100%, significantly enhancing precise genome editing capabilities. The increased HR rates enabled over 90% integration efficiency of triplex genes, as well as over 90% deletion rates of large DNA fragments up to 20 kb. Furthermore, using this developed CRISPR-Cpf1 system, triple genes were precisely integrated into the genome by one-step, enabling lycopene production in O. polymorpha. CONCLUSIONS: This novel multiplexed genome-editing tool mediated by CRISPR-Cpf1 can realize the deletion and integration of multiple genes, which holds great promise for accelerating engineering efforts on this non-conventional methylotrophic yeast for metabolic engineering and genomic evolution towards its application as an industrial cell factory.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.