Advancements and Obstacles of CRISPR-Cas9 Technology in Translational Research.
Ontology highlight
ABSTRACT:
SUBMITTER: You L
PROVIDER: S-EPMC6447755 | biostudies-literature | 2019 Jun
REPOSITORIES: biostudies-literature
Publications
Advancements and Obstacles of CRISPR-Cas9 Technology in Translational Research.
You Liting L Tong Ruizhan R Li Mengqian M Liu Yuncong Y Xue Jianxin J Lu You Y Lu You Y
Molecular therapy. Methods & clinical development 20190315
The expanding CRISPR-Cas9 technology is an easily accessible, programmable, and precise gene-editing tool with numerous applications, most notably in biomedical research. Together with advancements in genome and transcriptome sequencing in the era of metadata, genomic engineering with CRISPR-Cas9 meets the developmental requirements of precision medicine, and clinical tests using CRISPR-Cas9 are now possible. This review summarizes developments and established preclinical applications of CRISPR- ...[more]
Similar Datasets
Project description:The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.
| S-EPMC7171402 | biostudies-literature
Project description:This report is based on the Federation of American Societies for Experimental Biology's symposium, "Engaging basic Scientists in Translational Research: Identifying Opportunities, Overcoming Obstacles," held in Chevy Chase, MD, March 24-25, 2011. Meeting participants examined the benefits of engaging basic scientists in translational research, the challenges to their participation in translational research, and the roles that research institutions, funding organizations, professional societies, and scientific publishers can play to address these challenges.
| S-EPMC3419626 | biostudies-literature
Project description:Genetic analysis in model systems can provide a rich context for conceptual understanding of gene structure, regulation, and function. With an intent to create a rich learning experience in molecular genetics, we developed a semester-long course-based undergraduate research experience (CURE) using the CRISPR-Cas9 gene editing system to disrupt specific genes in the zebrafish. The course was offered to freshman students; nine students worked in four groups (two to three members per group) to design, synthesize, and test the nuclease activity of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/sgRNAs for targeted disruption of specific genes in the zebrafish. Each group worked with a gene with an already known mutant phenotype that can be visually scored and a gene that had not been studied in zebrafish previously. Embedded in the course were a series of workshop-styled units or tutorials, including tours to core facilities. The focus was on introducing and developing skills that could be accommodated within the span of a semester. Each group successfully cloned at least one plasmid-encoding CRISPR/sgRNA template, visually analyzed injected embryos, and performed genotyping assays to detect CRISPR-Cas9 activity. In-class discussions, a final end-of-semester written test, and group oral presentations were assessed for an understanding of the CRISPR-Cas9 system, application of the CRISPR-Cas9 system as a gene manipulation tool, and experimental methods used to create plasmid vectors and synthesize sgRNA. In addition, poster presentations were evaluated by faculty, graduate students, and senior undergraduate students at a University research exposition. Self-reflections in the form of group conversations were video recorded. All students (9/9) distinctly showed learning gains after completing the activity, but the extent of the gains was variable, as seen from results of a written test and poster presentation assessment. Qualitative analysis of evaluations and self-reporting data indicated several gains, suggesting that all students found many aspects of the CURE valuable and gained project-specific (conceptual) and transferrable skills (science process and science identity).
| S-EPMC5969413 | biostudies-literature
Project description:The CRISPR-Cas9 system functions in microbial viral pathogen recognition pathways by identifying and targeting foreign DNA for degradation. Recently, biotechnological advances have allowed scientists to use CRISPR-Cas9-based elements as a molecular tool to selectively modify DNA in a wide variety of other living systems. Given the emerging need to bring engaging CRISPR-Cas9 laboratory experiences to an undergraduate audience, we incorporated a CRISPR-based research project into our Genetics class laboratories, emphasizing its use in plants. Our genetic manipulations were designed for Arabidopsis thaliana, which despite serving as a plant research model, has traditionally been difficult to use in a classroom setting. For this project, students transformed plasmid DNA containing the essential CRISPR-Cas9 gene editing elements into A. thaliana. Expression of these elements in the plant genome was expected to create a deletion at one of six targeted genes. The genes we chose had a known seedling and/or juvenile loss-of-function phenotype, which made genetic analysis by students with a limited background possible. It also allowed the project to reach completion in a typical undergraduate semester timeframe. Assessment efforts demonstrated several learning gains, including students' understanding of CRISPR-Cas9 content, their ability to apply CRISPR-Cas9 gene editing tools using bioinformatics and genetics, their ability to employ elements of experimental design, and improved science communication skills. They also felt a stronger connection to their scientific education and were more likely to continue on a STEM career path. Overall, this project can be used to introduce CRISPR-Cas9 technology to undergraduates using plants in a single-semester laboratory course.
| S-EPMC6608611 | biostudies-literature
Project description:Despite their importance for most DNA-templated processes, the function of individual histone modifications has remained largely unknown because in vivo mutational analyses are lacking. The reason for this is that histone genes are encoded by multigene families and that tools to simultaneously edit multiple genomic loci with high efficiency are only now becoming available. To overcome these challenges, we have taken advantage of the power of CRISPR-Cas9 for precise genome editing and of the fact that most DNA repair in the protozoan parasite Trypanosoma brucei occurs via homologous recombination. By establishing an episome-based CRISPR-Cas9 system for T. brucei, we have edited wild type cells without inserting selectable markers, inserted a GFP tag between an ORF and its 3'UTR, deleted both alleles of a gene in a single transfection, and performed precise editing of genes that exist in multicopy arrays, replacing histone H4K4 with H4R4 in the absence of detectable off-target effects. The newly established genome editing toolbox allows for the generation of precise mutants without needing to change other regions of the genome, opening up opportunities to study the role of individual histone modifications, catalytic sites of enzymes or the regulatory potential of UTRs in their endogenous environments.
| S-EPMC6182134 | biostudies-literature
Project description:CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.
| S-EPMC6429391 | biostudies-literature
Project description:The mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA) binding protein essential for the initiation of transcription and genome maintenance. Recently it was demonstrated that the primary role of TFAM is to maintain the integrity of mtDNA and that it is a key regulator of mtDNA copy number. It was also shown that TFAM plays a central role in the mtDNA stress-mediated inflammatory response. In our study, we proposed to evaluate the possibility of editing the TFAM gene by CRISPR/Cas9 technology in bovine fibroblasts, as TFAM regulates the replication specificity of mtDNA. We further attempted to maintain these cells in culture post edition in a medium supplemented with uridine and pyruvate to mimic Rho zero cells that are capable of surviving without mtDNA, because it is known that the TFAM gene is lethal in knockout mice and chicken. Moreover, we evaluated the effects of TFAM modification on mtDNA copy number. The CRISPR gRNA was designed to target exon 1 of the bovine TFAM gene and subsequently cloned. Fibroblasts were transfected with Cas9 and control plasmids. After 24 h of transfection, cells were analyzed by flow cytometry to evaluate the efficiency of transfection. The site directed-mutation frequency was assessed by T7 endonuclease assay, and cell clones were analyzed for mtDNA copy number by Sanger DNA sequencing. We achieved transfection efficiency of 51.3%. We selected 23 successfully transformed clones for further analysis, and seven of these exhibited directed mutations at the CRISPR/Cas9 targeted site. Moreover, we also found a decrease in mtDNA copy number in the gene edited clones compared to that in the controls. These TFAM gene mutant cells were viable in culture when supplemented with uridine and pyruvate. We conclude that this CRISPR/Cas9 design was efficient, resulting in seven heterozygous mutant clones and opening up the possibility to use these mutant cell lines as a model system to elucidate the role of TFAM in the maintenance of mtDNA integrity.
| S-EPMC6405117 | biostudies-literature
Project description:Soybean is an important economic crop and a typical short-day crop, sensitive to photoperiod, and has narrow geographical adaptative region, which limit the creation of transgenic materials and reduce the breeding efficiency of new varieties. In addition, the genetic transformation efficiency of soybean is lower than that of many other crops, and the available receptor genotypes are limited. In this study, Agrobacterium-mediated transformation were used to introduce the CRISPR/Cas9 expression vector into soybean cultivar Jack and generated targeted mutants of E1 gene controlling soybean flowering. We obtained two novel types of mutations, 11 bp and 40 bp deletion at E1 coding region, respectively, and frameshift mutations produced premature translation termination codons and truncated E1 proteins, causing obvious early flowering under long day condition. In addition, no off-target effects were observed by predicting and analyzing the potential off-target sites of E1 targets. Significant decreased E1 gene expression of two novel mutants showed that the truncated E1 protein disinhibited GmFT2a/5a and increasing GmFT2a/5a gene expressions resulted obvious early flowering. Homozygous trans-clean mutants without T-DNA elements were also obtained and showed early flowering under long day condition. The photo-insensitive soybean transformation receptor we created laid a foundation for breeding excellent transgenic receptors suitable for high latitudes.
| S-EPMC6882952 | biostudies-literature
Project description:Massively parallel sequencing technologies have made it possible to generate large quantities of sequence data. However, as research-associated information is transferred into clinical practice, cost and throughput constraints generally require sequence-specific targeted analyses. Therefore, sample enrichment methods have been developed to meet the needs of clinical sequencing applications. However, current amplification and hybrid capture enrichment methods are limited in the contiguous length of sequences for which they are able to enrich. PCR based amplification also loses methylation data and other native DNA features. We have developed a novel technology (Negative Enrichment) where we demonstrate targeting long (>10 kb) genomic regions of interest. We use the specificity of CRISPR-Cas9 single guide RNA (Cas9/sgRNA) complexes to define 5' and 3' termini of sequence-specific loci in genomic DNA, targeting 10 to 36 kb regions. The complexes were found to provide protection from exonucleases, by protecting the targeted sequences from degradation, resulting in enriched, double-strand, non-amplified target sequences suitable for next-generation sequencing library preparation or other downstream analyses.
| S-EPMC6472885 | biostudies-literature
Project description:Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the expansion of CAG repeats in exon 1 of the huntingtin gene (HTT). Despite its monogenic nature, HD pathogenesis is still not fully understood, and no effective therapy is available to patients. The development of new techniques such as genome engineering has generated new opportunities in the field of disease modeling and enabled the generation of isogenic models with the same genetic background. These models are very valuable for studying the pathogenesis of a disease and for drug screening. Here, we report the generation of a series of homozygous HEK 293T cell lines with different numbers of CAG repeats at the HTT locus and demonstrate their usefulness for testing therapeutic reagents. In addition, using the CRISPR-Cas9 system, we corrected the mutation in HD human induced pluripotent stem cells and generated a knock-out of the HTT gene, thus providing a comprehensive set of isogenic cell lines for HD investigation.
| S-EPMC7084361 | biostudies-literature