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Genes Mar 2020Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination... (Review)
Review
Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.
Topics: Animals; CRISPR-Cas Systems; Gene Targeting; Gene Transfer Techniques; Genetic Engineering; Humans
PubMed: 32164255
DOI: 10.3390/genes11030291 -
Cell Jul 2013The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated...
The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.
Topics: Bacterial Proteins; Gene Targeting; HEK293 Cells; HeLa Cells; Humans; Saccharomyces cerevisiae; Streptococcus pyogenes; RNA, Small Untranslated
PubMed: 23849981
DOI: 10.1016/j.cell.2013.06.044 -
Methods in Molecular Biology (Clifton,... 2013The ability to introduce DNA sequences (e.g., genes) of interest into the germline genome has rendered the mouse a powerful and indispensable experimental model in... (Review)
Review
The ability to introduce DNA sequences (e.g., genes) of interest into the germline genome has rendered the mouse a powerful and indispensable experimental model in fundamental and medical research. The DNA sequences can be integrated into the genome randomly or into a specific locus by homologous recombination, in order to: (1) delete or insert mutations into genes of interest to determine their function, (2) introduce human genes into the genome of mice to generate animal models enabling study of human-specific genes and diseases, e.g., mice susceptible to infections by human-specific pathogens of interest, (3) introduce individual genes or genomes of pathogens (such as viruses) in order to examine the contributions of such genes to the pathogenesis of the parent pathogens, (4) and last but not least introduce reporter genes that allow monitoring in vivo or ex vivo the expression of genes of interest. Furthermore, the use of recombination systems, such as Cre/loxP or FRT/FLP, enables conditional induction or suppression of gene expression of interest in a restricted period of mouse's lifetime, in a particular cell type, or in a specific tissue. In this review, we will give an updated summary of the gene targeting technology and discuss some important considerations in the design of gene-targeted mice.
Topics: Animals; Cloning, Molecular; Embryonic Stem Cells; Gene Targeting; Genes, Reporter; Genetic Vectors; Homologous Recombination; Mice; Mice, Transgenic; Mutation; RNA, Untranslated; Transgenes; Viruses
PubMed: 23996268
DOI: 10.1007/978-1-62703-601-6_23 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2019The establishment and development of gene knockout mice have provided powerful support for the study of gene function and the treatment of human diseases. Gene targeting...
The establishment and development of gene knockout mice have provided powerful support for the study of gene function and the treatment of human diseases. Gene targeting and gene trap are two techniques for generating gene knockout mice from embryonic stem cells. Gene targeting replaces endogenous knockout gene by homologous recombination. There are two ways to knock out target genes: promoter trap and polyA trap. In recent years, many new gene knockout techniques have been developed, including Cre/loxP system, CRISP/Cas9 system, latest ZFN technology and TALEN technology. This article focuses on the several new knockout mouse techniques.
Topics: Animals; Disease Models, Animal; Embryonic Stem Cells; Gene Knockout Techniques; Gene Targeting; Homologous Recombination; Humans; Mice; Mice, Knockout
PubMed: 31222997
DOI: 10.13345/j.cjb.180417 -
Cell May 2013Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice...
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.
Topics: Animals; Base Sequence; Embryonic Stem Cells; Female; Gene Targeting; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Molecular Sequence Data; RNA, Small Untranslated
PubMed: 23643243
DOI: 10.1016/j.cell.2013.04.025 -
Nucleic Acids Research Nov 2016The CRISPR/Cas9 system is a powerful genome editing tool and has been widely used for biomedical research. However, many challenges, such as off-target effects and lack...
The CRISPR/Cas9 system is a powerful genome editing tool and has been widely used for biomedical research. However, many challenges, such as off-target effects and lack of easy solutions for multiplex targeting, are still limiting its applications. To overcome these challenges, we first developed a highly efficient doxycycline-inducible Cas9-EGFP vector. This vector allowed us to track the cells for uniform temporal control and efficient gene disruption, even in a polyclonal setting. Furthermore, the inducible CRISPR/Cas9 system dramatically decreased off-target effects with a pulse exposure of the genome to the Cas9/sgRNA complex. To target multiple genes simultaneously, we established simple one-step cloning approaches for expression of multiple sgRNAs with improved vectors. By combining our inducible and multiplex genome editing approaches, we were able to simultaneously delete Lysine Demethylase (KDM) 5A, 5B and 5C efficiently in vitro and in vivo This user friendly and highly efficient toolbox provides a solution for easy genome editing with tight temporal control, minimal off-target effects and multiplex targeting.
Topics: Bacterial Proteins; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Cell Line; Clustered Regularly Interspaced Short Palindromic Repeats; Endonucleases; Gene Expression; Gene Knockout Techniques; Gene Order; Gene Silencing; Gene Targeting; Genes, Reporter; Genetic Vectors; Humans; Promoter Regions, Genetic; RNA, Guide, CRISPR-Cas Systems; Retinoblastoma-Binding Protein 2
PubMed: 27458201
DOI: 10.1093/nar/gkw660 -
Journal of Hepatology Feb 2022Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. The advanced stage of NAFLD, non-alcoholic steatohepatitis (NASH),...
BACKGROUND & AIMS
Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. The advanced stage of NAFLD, non-alcoholic steatohepatitis (NASH), has been recognized as a leading cause of end-stage liver injury for which there are no FDA-approved therapeutic options. Glutathione S-transferase Mu 2 (GSTM2) is a phase II detoxification enzyme. However, the roles of GSTM2 in NASH have not been elucidated.
METHODS
Multiple RNA-seq analyses were used to identify hepatic GSTM2 expression in NASH. In vitro and in vivo gain- or loss-of-function approaches were used to investigate the role and molecular mechanism of GSTM2 in NASH.
RESULTS
We identified GSTM2 as a sensitive responder and effective suppressor of NASH progression. GSTM2 was significantly downregulated during NASH progression. Hepatocyte GSTM2 deficiency markedly aggravated insulin resistance, hepatic steatosis, inflammation and fibrosis induced by a high-fat diet and a high-fat/high-cholesterol diet. Mechanistically, GSTM2 sustained MAPK pathway signaling by directly interacting with apoptosis signal-regulating kinase 1 (ASK1). GSTM2 directly bound to the N-terminal region of ASK1 and inhibited ASK1 N-terminal dimerization to subsequently repress ASK1 phosphorylation and the activation of its downstream JNK/p38 signaling pathway under conditions of metabolic dysfunction.
CONCLUSIONS
These data demonstrated that hepatocyte GSTM2 is an endogenous suppressor that protects against NASH progression by blocking ASK1 N-terminal dimerization and phosphorylation. Activating GSTM2 holds promise as a therapeutic strategy for NASH.
CLINICAL TRIAL NUMBER
IIT-2021-277.
LAY SUMMARY
New therapeutic strategies for non-alcoholic steatohepatitis are urgently needed. We identified that the protein GSTM2 exerts a protective effect in response to metabolic stress. Therapies that aim to increase the activity of GSTM2 could hold promise for the treatment of non-alcoholic steatohepatitis.
Topics: Animals; Biopsy; Disease Models, Animal; Gene Targeting; Glutathione Transferase; Hepatocytes; Liver; MAP Kinase Kinase Kinase 5; Mice; Non-alcoholic Fatty Liver Disease; Sequence Analysis, RNA
PubMed: 34656650
DOI: 10.1016/j.jhep.2021.09.040 -
Molecular Plant Aug 2020CRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic deletions. However, precise gene insertion or sequence replacement remains a major hurdle...
CRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic deletions. However, precise gene insertion or sequence replacement remains a major hurdle before application of CRISPR-Cas9 technology is fully realized in plant breeding. Here, we report high-frequency, selectable marker-free intra-genomic gene targeting (GT) in maize. Heat shock-inducible Cas9 was used for generating targeted double-strand breaks and simultaneous mobilization of the donor template from pre-integrated T-DNA. The construct was designed such that release of the donor template and subsequent DNA repair activated expression of the selectable marker gene within the donor locus. This approach generated up to 4.7% targeted insertion of the donor sequence into the target locus in T0 plants, with up to 86% detected donor template release and 99% mutation rate being observed at the donor loci and the genomic target site, respectively. Unlike previous in planta or intra-genomic homologous recombination reports in which the original chimeric GT plants required extensive progeny screening in the next generation to identify non-chimeric GT individuals, our method provides non-chimeric heritable GT in one generation.
Topics: CRISPR-Cas Systems; Gene Targeting; Genetic Markers; Kanamycin Kinase; Mutagenesis, Insertional; Zea mays
PubMed: 32574856
DOI: 10.1016/j.molp.2020.06.008 -
Biotechnology Letters Jun 2016To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal...
OBJECTIVES
To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection.
RESULTS
Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells.
CONCLUSION
Lipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.
Topics: Animals; CRISPR-Cas Systems; Cell Line; Electroporation; Gene Editing; Gene Targeting; Green Fluorescent Proteins; Humans; Induced Pluripotent Stem Cells; Lipids; Mice; Transfection
PubMed: 26892225
DOI: 10.1007/s10529-016-2064-9 -
Nature Reviews. Genetics Sep 2015Next-generation DNA sequencing has revealed the complete genome sequences of numerous organisms, establishing a fundamental and growing understanding of genetic... (Review)
Review
Next-generation DNA sequencing has revealed the complete genome sequences of numerous organisms, establishing a fundamental and growing understanding of genetic variation and phenotypic diversity. Engineering at the gene, network and whole-genome scale aims to introduce targeted genetic changes both to explore emergent phenotypes and to introduce new functionalities. Expansion of these approaches into massively parallel platforms establishes the ability to generate targeted genome modifications, elucidating causal links between genotype and phenotype, as well as the ability to design and reprogramme organisms. In this Review, we explore techniques and applications in genome engineering, outlining key advances and defining challenges.
Topics: Animals; Gene Targeting; Genetic Engineering; Genetic Variation; Genome; Genotype; High-Throughput Nucleotide Sequencing; Humans
PubMed: 26260262
DOI: 10.1038/nrg3956