scholarly journals The Potential Use of the CRISPR-Cas System for HIV-1 Gene Therapy

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Gabriela De Nardi Sanches-da-Silva ◽  
Luiza Fonseca Sales Medeiros ◽  
Fabio Mitsuo Lima
Keyword(s):  
Gene Therapy ◽  
Current Knowledge ◽  
Genetic Material ◽  
Specific Gene ◽  
Loss Of Function ◽  
Complementary Sequence ◽  
Guide Rna ◽  
Ethical Implications ◽  
A Genome ◽  
Hiv 1

The HIV-1 virus (human immunodeficiency virus) affects 36.9 million people worldwide, with approximately 900000 deaths in 2017. The virus carrier can develop severe immunodeficiency since CD4+ T lymphocytes are the main target, leading to acquired immunodeficiency syndrome (AIDS). Despite advances in pharmacological treatment, it is still difficult to eliminate latent reservoirs, becoming one of the main obstacles for viral eradication. The CRISPR- (clustered regularly interspaced short palindromic repeat-) Cas system is a genome-editing method which uses a guide RNA, a complementary sequence to the interested site, recruiting a nuclease that can break the viral or the host cell genetic material. From this double-stranded break, cellular repair mechanisms are activated being able to generate deletions, insertions, or substitutions, in order to inactivate specific gene loci, leading to loss of function. The objective of this minireview is to synthesize the current knowledge on the application of CRISPR-Cas-based gene therapy for HIV-1. The strategies encompass all steps of the viral infection cycle, from inhibition of cell invasion, through viral replication and integration inhibition, to excision of the latent provirus. Off-target effects and ethical implications were also discussed to evaluate the safety of the approach and viability of its application in humans, respectively. Although preclinical and clinical tests are still needed, the recent results establish an exciting possibility of applying this technology for prophylaxis and treatment of HIV-1.

scholarly journals Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs

2018 ◽  
Vol 115 (40) ◽  
pp. E9343-E9352 ◽  
Author(s):  
Georg Oberhofer ◽  
Tobin Ivy ◽  
Bruce A. Hay
Keyword(s):  
Homing Endonuclease ◽  
Marker Gene ◽  
Specific Gene ◽  
Gene Drive ◽  
Loss Of Function ◽  
End Joining ◽  
Complex Activity ◽  
Guide Rna ◽  
Type Gene ◽  
Population Suppression

A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work inDrosophilaand mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs inDrosophila melanogastertargeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.

scholarly journals Genetic Screens in Human Cells Using the CRISPR-Cas9 System

Science ◽  
2013 ◽  
Vol 343 (6166) ◽  
pp. 80-84 ◽  
Author(s):  
Tim Wang ◽  
Jenny J. Wei ◽  
David M. Sabatini ◽  
Eric S. Lander
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Negative Selection ◽  
Topoisomerase Ii ◽  
Massively Parallel Sequencing ◽  
Sequence Motifs ◽  
Loss Of Function ◽  
Specific Sequence ◽  
Guide Rna ◽  
A Genome

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

CRISPR/Cas9 genome editing technology applications in biological and biomedical fields

2021 ◽  
Author(s):  
Moataz Dowaidar
Keyword(s):  
Gene Therapy ◽  
Genome Editing ◽  
Dna Sequences ◽  
Recombinant Dna ◽  
Genetic Disorders ◽  
Specific Gene ◽  
Recombinant Dna Technology ◽  
P Gene ◽  
A Genome ◽  
Biomedical Fields

Gene therapy is a way of mending or replacing a gene in an undesirable or non-functional cell. Although used in both animals and plants, gene therapy is most usually linked with humans. Because there are so many genetic disorders caused by genetic abnormalities or unwanted gene expression, gene therapy is promising to treat and even cure many diseases. The scientific and pharmaceutical sectors are becoming interested in gene therapy.CRISPR was initially detected in prokaryotic organisms, bacteria and archaea genomes. Although nucleotide sequences are regularly discovered in many bacteria and archaea, the scientific community has not realized its importance for over a decade. People used these diverse DNA sequences as a diagnostic for genotyping and therefore considered them as a distinctive feature for each particular microbe. Scientists are beginning to comprehend that the CRISPR/Cas system is a prokaryotic defense system's adaptive immunity to viruses, due to the discovery of CRISPR-associated protein (Cas) and the use of recombinant DNA technology. This recently discovered CRISPR/Cas system was swiftly developed as a tool for editing a specific gene in a genome. Since 2012, CRISPR/Cas9 genome editing technology has been quickly researched and applied in several biological and biomedical fields. For various basic and practical research reasons, as well as biotechnological applications in agriculture and healthcare, CRISPR/Cas9 technology has altered and improved greatly over the past five years. Base editor invention and prime editing technology by fusing a Cas endonuclease with other functional enzymes, such as base converter enzymes, is one of several milestones in this fast progress.

scholarly journals Highly efficient CRISPR/Cas9-mediated tissue specific mutagenesis in Drosophila

2018 ◽  
Author(s):  
Amy R. Poe ◽  
Bei Wang ◽  
Maria L. Sapar ◽  
Hui Ji ◽  
Kailyn Li ◽  
...  
Keyword(s):  
Gene Function ◽  
Gene Knockout ◽  
Somatic Cells ◽  
Specific Gene ◽  
Loss Of Function ◽  
Neuronal Morphogenesis ◽  
Tissue Specific ◽  
Guide Rna ◽  
Tissue Specific Gene ◽  
Highly Efficient

ABSTRACTTissue-specific loss-of-function (LOF) analysis is an essential approach for characterizing gene function. Here we describe an efficient CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila. This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. To facilitate the construction of these components, we developed convenient tools for generating and evaluating enhancer-driven Cas9 lines, identified a multi-gRNA design that is highly efficient in mutagenizing somatic cells, and established an assay for testing the efficiency of multi-gRNAs in creating double-stranded breaks. We found that excision of genomic DNA induced by two gRNAs is infrequent in somatic cells, while indels more reliably cause tissue-specific LOF. Furthermore, we show that enhancer-driven Cas9 is less cytotoxic yet results in more complete gene removal than Gal4-driven Cas9 in larval neurons. Finally, we demonstrate that CRISPR-TRiM efficiently unmasks redundant gene functions in neuronal morphogenesis. Importantly, two Cas9 transgenes that turn on with different timings in the neuronal lineage revealed the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISRPR tools can be applied to analyze tissue-specific gene function in many biological processes.

Association of Micro RNA and Postoperative Cognitive Dysfunction: A Review

2020 ◽  
Vol 20 (17) ◽  
pp. 1781-1790
Author(s):  
Noor Anisah Abu Yazit ◽  
Norsham Juliana ◽  
Srijit Das ◽  
Nur Islami Mohd Fahmi Teng ◽  
Nadia Mohd Fahmy ◽  
...  
Keyword(s):  
Cognitive Dysfunction ◽  
Chromosomal Abnormalities ◽  
Current Knowledge ◽  
System Development ◽  
Genetic Disorder ◽  
Postoperative Cognitive Dysfunction ◽  
Clinical Importance ◽  
Nervous System Development ◽  
Micro Rna ◽  
A Genome

Postoperative Cognitive Dysfunction (POCD) refers to the condition of neurocognitive decline following surgery in a cognitive and sensory manner. There are several risk factors, which may be life-threatening for this condition. Neuropsychological assessment of this condition is very important. In the present review, we discuss the association of apolipoprotein epsilon 4 (APOE ε4) and few miRNAs with POCD, and highlight the clinical importance for prognosis, diagnosis and treatment of POCD. Microarray is a genome analysis that can be used to determine DNA abnormalities. This current technique is rapid, efficient and high-throughout. Microarray techniques are widely used to diagnose diseases, particularly in genetic disorder, chromosomal abnormalities, mutations, infectious diseases and disease-relevant biomarkers. MicroRNAs (miRNAs) are a class of non-coding RNAs that are widely found distributed in eukaryotes. Few miRNAs influence the nervous system development, and nerve damage repair. Microarray approach can be utilized to understand the miRNAs involved and their pathways in POCD development, unleashing their potential to be considered as a diagnostic marker for POCD. This paper summarizes and identifies the studies that use microarray based approaches for POCD analysis. Since the application of microarray in POCD is expanding, there is a need to review the current knowledge of this approach.

scholarly journals How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

2021 ◽  
Vol 22 (14) ◽  
pp. 7545
Author(s):  
Myriam Sainz-Ramos ◽  
Idoia Gallego ◽  
Ilia Villate-Beitia ◽  
Jon Zarate ◽  
Iván Maldonado ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Clinical Practice ◽  
Gene Delivery ◽  
Viral Vectors ◽  
Viral Vector ◽  
Clinical Success ◽  
Genetic Material ◽  
Viral Gene ◽  
Promising Alternative ◽  
Vector Systems

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.

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