Your browser does not allow saving data (e.g. private mode): bookmarks will be lost when the tab closes.
Search PubMed
Keywords, author, journal, years — results from PubMed, abstracts with Russian translation, bookmarks in your browser.
Found: 26 075
Page 70 of 1000-
CRISPRing the Elephant in the Room.
The importance of guanylyl-cyclases (GCs) in apicomplexa has remained elusive due to the large size of the genes. Two recent studies, including Brown and Sibley, 2018 in this issue of Cell Host & Microbe, make elegant use of genome editing with CRISPR-Cas9 to characterize roles of GCs in Toxoplasma and Plasmodium.
-
CRISPR Genome Editing Technology and its Application in Genetic Diseases: A Review.
Gene therapy has been a long lasting goal for scientists, and there are many optimal methods and tools to correct disease-causing mutations in humans. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been progressively adopted for the assessment a treatment of human diseases, including thalassemia, Parkinson's disease, cystic fibrosis, glaucoma, Huntington's disease, and Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS). CRISPR sequences belong to the bacterial immune system, which includes the nuclease Cas enzyme and an RNA sequence. The RNA sequence is unique and pathogen-specific, and identifies and binds to the DNA of invasive viruses, allowing the nuclease Cas enzyme to cut the identified DNA and destroy the invasive viruses. This feature provides the possibility to edit mutations in the DNA sequence of live cells by replacing a specific targeted RNA sequence with the RNA sequence in the CRISPR system. Previous studies have reported the improvement steps in confrontation with human diseases caused by single-nucleotide mutations using this system. In this review, we first introduce CRISPR and its functions and then elaborate on the use of CRISPR in the treatment of human diseases.
-
Fueling chromosomal gene diversification and artificial evolution with CRISPR.
Gene diversification is an effective approach to massively dissecting variant functions and evolving sequences when paired with an appropriate assay. In vitro mutagenesis and ectopic gene expression, however, fail to simulate the endogenous regulatory environment of the variants. The development of clustered, regularly interspaced short palindromic repeats (CRISPR) systems has greatly boosted the efficiency of targeted gene diversification in various species. Here, we review recent CRISPR-assisted methods for chromosomal gene diversification and artificial evolution, focusing on the advantages and limitations of each approach, and propose possible strategies to overcome current limitations and directions in future technology development.
-
[The research advances and applications of genome editing in hereditary eye diseases].
Genome editing is a cutting-edge technology that generates DNA double strand breaks at the specific genomic DNA sequence through nuclease recognition and cleavage, and then achieves insertion, replacement, or deletion of the target gene via endogenous DNA repair mechanisms, such as non-homologous end joining, homology directed repair, and homologous recombination. So far, more than 600 human hereditary eye diseases and systemic hereditary diseases with ocular phenotypes have been found. However, most of these diseases are of incompletely elucidated pathogenesis and without effective therapies. Genome editing technology can precisely target and alter the genomes of animals, establish animal models of the hereditary diseases, and elucidate the relationship between the target gene and the disease phenotype, thereby providing a powerful approach to studying the pathogenic mechanisms underlying the hereditary eye diseases. In addition, correction of gene mutations by the genome editing brings a new hope to gene therapy for the hereditary eye diseases. This review introduces the molecular characteristics of 4 major enzymes used in the genome editing, including homing endonucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9), and summarizes the current applications of this technology in investigating the pathogenic mechanisms underlying the hereditary eye diseases. (Chin J Ophthalmol, 2017, 53: 386-371).
-
CRISPR-cas System as a Genome Engineering Platform: Applications in Biomedicine and Biotechnology.
Genome editing mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated proteins (Cas) has recently been considered to be used as efficient, rapid and site-specific tool in the modification of endogenous genes in biomedically important cell types and whole organisms. It has become a predictable and precise method of choice for genome engineering by specifying a 20-nt targeting sequence within its guide RNA. Firstly, this review aims to describe the biology of CRISPR system. Next, the applications of CRISPR-Cas9 in various ways, such as efficient generation of a wide variety of biomedically important cellular models as well as those of animals, modifying epigenomes, conducting genome-wide screens, gene therapy, labelling specific genomic loci in living cells, metabolic engineering of yeast and bacteria and endogenous gene expression regulation by an altered version of this system were reviewed.
-
Chemically Modified Cpf1-CRISPR RNAs Mediate Efficient Genome Editing in Mammalian Cells.
CRISPR-based gene editing is a powerful technology for engineering mammalian genomes. It holds the potential as a therapeutic, although much-needed in vivo delivery systems have yet to be established. Here, using the Cpf1-crRNA (CRISPR RNA) crystal structure as a guide, we synthesized a series of systematically truncated and chemically modified crRNAs, and identify positions that are amenable to modification while retaining gene-editing activity. Modified crRNAs were designed with the same modifications that provide protection against nucleases and enable wide distribution in vivo. We show crRNAs with chemically modified terminal nucleotides are exonuclease resistant while retaining gene-editing activity. Chemically modified or DNA-substituted nucleotides at select positions and up to 70% of the crRNA DNA specificity region are also well tolerated. In addition, gene-editing activity is maintained with phosphorothioate backbone substitutions in the crRNA DNA specificity region. Finally, we demonstrate that 42-mer synthetic crRNAs from the similar CRISPR-Cas9 system are taken up by cells, an attractive property for in vivo delivery. Our study is the first to show that chemically modified crRNAs of the CRISPR-Cpf1 system can functionally replace and mediate comparable gene editing to the natural crRNA, which holds the potential for enhancing both viral- and non-viral-mediated in vivo gene editing.
-
New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing.
CRISPR-Cas has proven to be the most versatile genetic tinkering system of our time, predominantly as a precision genome editing tool. Here, we demonstrate two additions to the repertoire of CRISPR's application for constructing donor DNA templates: CRISPR-CLONInG and CRISPR-CLIP. CRISPR-CLONInG (CRISPR-Cutting and Ligation Of Nucleic acid In vitro via Gibson) was devised to enable efficient cut-and-paste of multiple complex DNA fragments by using CRISPR-Cas9 as a digestion alternative with precision and exclusivity features, followed by joining the digested products via Gibson Assembly, to construct double-stranded DNA and adeno-associated virus (AAV) donor vectors rapidly without cloning scars. CRISPR-CLIP (CRISPR-Clipped Long ssDNA via Incising Plasmid) was devised as a DNA clipping tool to retrieve long single-stranded DNA (lssDNA) efficiently from plasmid, up to 3.5 kbase, which can be supplied as the donor template for creating genetically engineered mice via Easi-CRISPR. We utilized two different Cas types (Cpf1 and Cas9n) to induce two distinct incisions at the respective ends of the lssDNA cassette junctions on the plasmid, yielding three independent single-stranded DNA units of unique sizes eligible for strand separation, followed by target strand clip-out through gel extraction. The retrieval of the lssDNA donor circumvents involvements of restriction enzymes and DNA polymerase-based steps. Hence, it not only retains sequence fidelity but also carries virtually no restriction on sequence composition, further mitigating limitations on the current Easi-CRISPR method. With the add-on feature of universal DNA-tag sequences of Cpf1-Cas9 duo protospacer adjacent motif, CRISPR-CLIP can be facile and applicable to generate lssDNA templates for any genomic target of choice. Additionally, we demonstrate robust gene editing efficiencies in the neuroblastoma cell line, as well as in mice attained with the AAV and lssDNA donors constructed herein.
-
The Promise of CRISPR for Human Germline Editing and the Perils of "Playing God".
In the midst of the media and professional exuberance regarding the potential benefits of CRISPR technology, voices of criticism and caution have also arisen. One of the thorniest such cautions has been the common objection that CRISPR allows bioscientists to "play God," particularly when it comes to potentially editing the human germline. Many in the biotechnology field are unsure how to address this concern. What does it mean, particularly for bioscientists who may not have any rational or rhetorical categories for God? In this article, I explore possible meanings of "playing God" and the arguments for how those meanings might be applied in the utilization of CRISPR technology for human germline editing. I then test the validity of those arguments and explore potential counterarguments. Finally, I discuss how members of the bioscience community might respond to the objection of "playing God" and contribute to that dialogue in ways that could impact the future of CRISPR development and applications.
-
Synthetic Oligonucleotides Inhibit CRISPR-Cpf1-Mediated Genome Editing.
Previously, researchers discovered a series of anti-CRISPR proteins that inhibit CRISPR-Cas activity, such as Cas9 and Cpf1 (Cas12a). Herein, we constructed crRNA variants consisting of chemically modified DNA-crRNA and RNA-crRNA duplexes and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1. More important, without prehybridization, these PS-modified DNA oligonucleotides showed the ability to suppress DNA double-strand breaks induced by two Cpf1 orthologs, AsCpf1 and LbCpf1. Time-dependent inhibitory effects were validated in multiple loci of different human cells. Further studies demonstrated that PS-modified DNA oligonucleotides were able to serve as Cpf1 inhibitors in a sequence-independent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder target DNA binding and recognition by Cpf1. Consequently, these synthetic DNA molecules expand the sources of CRISPR inhibitors, providing a platform to inactivate Cpf1-mediated genome editing.
-
Leveraging advances in RNAi and CRISPR for improved biological pest control.
The limitations of chemical pesticides and their associated risks highlight the need for more sustainable pest management strategies. Biological control using natural enemies offers an eco-friendly alternative but is sometimes constrained by efficiency and scalability. Emerging molecular tools-RNA interference (RNAi) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene editing-present novel opportunities to enhance existing biological control or to control pests directly. RNAi induces targeted gene knockdown via a non-heritable, transient response. CRISPR enables precise genetic modifications and could improve traits in beneficial insects or disrupt essential genes in pests, optionally including a gene drive for increased power. Although limitations remain for several species, these technologies could be valuable tools for integrated pest management. Their future implementation raises biosafety and regulatory considerations, particularly for self-propagating systems like gene drives. This review showcases developments in RNAi and CRISPR-based pest control, and calls for risk-based, adaptive governance to enable their responsible use in sustainable agriculture.
PubMed returns only the first 10,000 results — refine the query to see the rest.