Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 63
Filter
1.
Electron. j. biotechnol ; 52: 59-66, July. 2021. ilus, tab
Article in English | LILACS | ID: biblio-1283592

ABSTRACT

BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.


Subject(s)
Animals , Mice , Adenosine Deaminase , Cytosine , CRISPR-Cas Systems , Gene Editing/methods , Base Sequence , Blotting, Western , Models, Animal , Real-Time Polymerase Chain Reaction , Mutation
2.
Chinese Journal of Biotechnology ; (12): 2307-2321, 2021.
Article in Chinese | WPRIM | ID: wpr-887798

ABSTRACT

The CRISPR system is able to accomplish precise base editing in genomic DNA, but relies on the cellular homology-directed recombination repair pathway and is therefore extremely inefficient. Base editing is a new genome editing technique developed based on the CRISPR/Cas9 system. Two base editors (cytosine base editor and adenine base editor) were developed by fusing catalytically disabled nucleases with different necleobase deaminases. These two base editors are able to perform C>T (G>A) or A>G (T>C) transition without generating DNA double-stranded breaks. The base editing technique has been widely used in gene therapy, animal models construction, precision animal breeding and gene function analysis, providing a powerful tool for basic and applied research. This review summarized the development process, technical advantages, current applications, challenges and perspectives for base editing technique, aiming to help the readers better understand and use the base editing technique.


Subject(s)
Adenine , Animals , CRISPR-Cas Systems/genetics , Cytosine , DNA Breaks, Double-Stranded , Gene Editing
3.
Journal of Experimental Hematology ; (6): 1676-1679, 2021.
Article in Chinese | WPRIM | ID: wpr-922316

ABSTRACT

β-thalassemia is a monogenetic inherited hemolytic anemia, which results in a series of pathophysiological changes due to partial or complete inhibition of the synthesis of β-globin chain. The curative therapy for this disease is to reconstitute hematopoiesis, and transplantation with genetically modified autologous hematopoietic stem cells can avoid the major difficulties of traditional allogeneic hematopoietic stem cell transplantation,such as HLA matching and immune rejection. β-thalassemia gene therapy strategies mainly include gene integration and genome editing. The former relies on the development of lentiviral vectors and adds a fully functional HBB gene to the chromosome; the latter rapidly develops with the research of specific nuclease which can repair the HBB gene in situ. In this review, the latest progress of the two strategies in gene therapy of β-thalassemia is summarized.


Subject(s)
Gene Editing , Genetic Therapy , Genetic Vectors , Humans , beta-Globins/genetics , beta-Thalassemia/therapy
4.
Chinese Journal of Biotechnology ; (12): 4342-4350, 2021.
Article in Chinese | WPRIM | ID: wpr-921510

ABSTRACT

The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.


Subject(s)
Animals , Bombyx/metabolism , CRISPR-Cas Systems/genetics , Endonucleases/genetics , Gene Editing , RNA, Guide/genetics
5.
Chinese Journal of Biotechnology ; (12): 3961-3974, 2021.
Article in Chinese | WPRIM | ID: wpr-921479

ABSTRACT

Novel model systems have provided powerful tools for the research of human biology. Despite of being widely used, the conventional research models could not precisely describe the human physiological phenomenon. Organoids are three-dimensional multicellular aggregates derived from stem cells or organ progenitors that could differentiate and self-organize to recapitulate some specific functionalities and architectures of their in vivo counterpart organs. Organoids can be used to simulate organogenesis because of their human origin. In addition, the genomic stability of organoids could be well maintained during long-term amplification in vitro. Moreover, organoids can be cryopreserved as a live biobank for high-throughput screening. Combinatorial use of organoids with other emerging technologies (e.g. gene editing, organ-on-a-chip and single-cell RNA sequencing) could overcome the bottlenecks of conventional models and provide valuable information for disease modelling, pharmaceutical research, precision medicine and regenerative medicine at the organ level. This review summarizes the classifications, characteristics, current applications, combined use with other technologies and future prospects of organoids.


Subject(s)
Gene Editing , Humans , Models, Biological , Organoids , Regenerative Medicine , Stem Cells
6.
Chinese Journal of Biotechnology ; (12): 3880-3889, 2021.
Article in Chinese | WPRIM | ID: wpr-921473

ABSTRACT

In the application of CRISPR genome editing, direct cellular delivery of non-replicable Cas9/sgRNA may reduce unwanted gene targeting and integrational mutagenesis, thus offering greater specificity and safety. Cas9/sgRNA delivery system holds great potential for treating genetic diseases. This review summarizes the advances of Cas9/sgRNA delivery systems and its therapeutic applications, providing new understandings and inspirations for vector design and future clinical applications.


Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing , RNA, Guide/genetics
7.
Chinese Journal of Biotechnology ; (12): 3071-3087, 2021.
Article in Chinese | WPRIM | ID: wpr-921407

ABSTRACT

In recent years, the genome editing technologies based on the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) have developed rapidly. The system can use homologous directed recombination (HDR) to achieve precise editing that it medicated, but the efficiency is extremely low, which limits its application in agriculture and biomedical fields. As an emerging genome editing technology, the CRISPR/Cas-mediated DNA base editing technologies can achieve targeted mutations of bases without generating double-strand breaks, and has higher editing efficiency and specificity compared with CRISPR/Cas-mediated HDR editing. At present, cytidine base editors (CBEs) that can mutate C to T, adenine base editors (ABEs) that can mutate A to G, and prime editors (PEs) that enable arbitrary base conversion and precise insertion and deletion of small fragments, have been developed. In addition, glycosylase base editors (GBEs) capable of transitioning from C to G and double base editors capable of editing both A and C simultaneously, have been developed. This review summarizes the development, advances, advantages and limitations of several DNA base editors. The successful applications of DNA base editing technology in biomedicine and agriculture, together with the prospects for further optimization and selection of DNA base editors, are discussed.


Subject(s)
Agriculture , CRISPR-Cas Systems/genetics , DNA/genetics , Gene Editing , Technology
8.
Chinese Journal of Biotechnology ; (12): 3061-3070, 2021.
Article in Chinese | WPRIM | ID: wpr-921406

ABSTRACT

The study of distinct genes, chromosomes and the spatio-temporal relationships between them is of great significance in genetics, developmental biology and biomedicine. CRISPR/Cas9 has become the most widely used gene editing tool due to its excellent targeting ability. Recently, researchers have developed a series of advanced live cell imaging techniques based on the nuclease-inactivated mutant of Cas9 (dCas9), providing rapid and convenient tools for high-resolution imaging of specific sites in the chromatin and genome. This review summarizes the advances of CRISPR/dCas9 system in live cell imaging from three aspects, including the strategies of cell delivery, optimization of the fluorescence signals, as well as orthogonal and multicolor imaging. Furthermore, we shed light on the development trends and prospects of this field.


Subject(s)
CRISPR-Cas Systems/genetics , Chromatin , Endonucleases , Gene Editing
9.
Acta Physiologica Sinica ; (6): 694-706, 2021.
Article in Chinese | WPRIM | ID: wpr-921273

ABSTRACT

The high failure rate of the new drug development has been well recognized. Relying on the pre-clinical data obtained from animal experiments will inevitably cause a low concordance with human clinical trials, which will eventually lead to new drug development failure. Employing human induced pluripotent stem cells (iPSCs) or adult stem cells to simulate disease models can not only provide an unlimited cell materials, but also faithfully represent the genetic background of a certain disease, when iPSCs or adult stem cells derived from patients with a specific disease genetic variation are applied. In addition, gene editing methods can be used to introduce genetic variants of interest into stem cells to generate disease models. Furthermore, by establishing a cell bank with a population of iPSCs in petri dish, in vitro human genetic studies can be carried out in these cells, with GWAS and QTL studies applied to identify genetic variants that are associated with drug sensitivity or cytotoxicity. These efforts may offer valuable information for the recruitment of suitable patients for clinical trials. Therefore, stem cell-derived disease models can provide valuable resources for the pathophysiological studies of diseases as well as the drug development. In this review, we will briefly introduce the development of the liver disease models derived from stem cells and their applications in disease study and drug development.


Subject(s)
Animals , Cell Differentiation , Drug Development , Gene Editing , Humans , Induced Pluripotent Stem Cells , Liver
10.
Article in Chinese | WPRIM | ID: wpr-880079

ABSTRACT

OBJECTIVE@#To identify differentiation related miRNA and evaluate roles of miRNA during ATRA induced myeloid differentiation.@*METHODS@#The small RNA sequencing was used to analyze differential expressed miRNAs in ATRA induced NB4 cells. Then the several up or down-regulated miRNA were selected as the research candidates. SgRNAs targeting the genome of each miRNA were designed and NB4 cells with inducible expression of Cas9 protein were generated. After transduced sgRNA into NB4/Cas9 cells, the mutation level by PCR and surveyor assay were evaluated. The cell differentiation level was investigated by surface CD11b expression via flow cytometry.@*RESULTS@#A total of 410 mature miRNAs which expressed in NB4 cells were detected out after treated by ATRA, 74 miRNAs were up-regulated and 55 were down-regulated miRNAs with DNA cleavage generated by CRISPR/Cas9 was assayed directly by PCR or surveyor assay, quantitative PCR showed that the expression of miRNA was downregulated, which evaluated that gene edition successfully inhibitied the expression of mature miRNA. MiR-223 knockout showed the myeloid differentation of NB4 significantly inhibitied, while miRNA-155 knockout showed the myeloid differentation of NB4 cells significantly increased.@*CONCLUSION@#CRISPR/Cas9 is a powerful tool for gene editing and can lead to miRNA knockout. Knockouts of miR-223 and miR-155 have shown a differentiation-related phenotype, and the potential mechanism is the integrative regulation of target genes.


Subject(s)
CRISPR-Cas Systems , Cell Differentiation , Gene Editing , MicroRNAs/genetics , Sequence Analysis, RNA , Tretinoin
11.
Article in Chinese | WPRIM | ID: wpr-879256

ABSTRACT

The emergence of regular short repetitive palindromic sequence clusters (CRISPR) and CRISPR- associated proteins 9 (Cas9) gene editing technology has greatly promoted the wide application of genetically modified pigs. Efficient single guide RNA (sgRNA) is the key to the success of gene editing using CRISPR/Cas9 technology. For large animals with a long reproductive cycle, such as pigs, it is necessary to screen out efficient sgRNA


Subject(s)
Animals , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing , RNA, Guide/genetics , Swine
12.
Chinese Journal of Biotechnology ; (12): 1659-1676, 2021.
Article in Chinese | WPRIM | ID: wpr-878660

ABSTRACT

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.


Subject(s)
Gene Editing , Metabolic Engineering , Pichia/genetics , Synthetic Biology , Yarrowia/genetics , Yeasts
13.
Chinese Journal of Biotechnology ; (12): 1619-1636, 2021.
Article in Chinese | WPRIM | ID: wpr-878658

ABSTRACT

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Gene Editing , Metabolic Engineering , Promoter Regions, Genetic
14.
Chinese Journal of Biotechnology ; (12): 1603-1618, 2021.
Article in Chinese | WPRIM | ID: wpr-878657

ABSTRACT

Corynebacterium glutamicum is an important workhorse of industrial biotechnology, especially for amino acid bioindustry. This bacterium is being used to produce various amino acids at a level of over 6 million tons per year. In recent years, enabling technologies for C. glutamicum metabolic engineering have been developed and improved, which accelerated construction and optimization of microbial cell factoriers, expanding spectra of substrates and products, and facilitated basic researches on C. glutamicum. With these technologies, C. glutamicum has become one of the ideal microbial chasses. This review summarizes recent key technological developments of enabling technologies for C. glutamicum metabolic engineering and focuses on establishment and applications of CRISPR-based genome editing, gene expression regulation, adaptive laboratory evolution, and biosensor technologies.


Subject(s)
Amino Acids , Biotechnology , Corynebacterium glutamicum/genetics , Gene Editing , Metabolic Engineering
15.
Chinese Journal of Biotechnology ; (12): 1564-1577, 2021.
Article in Chinese | WPRIM | ID: wpr-878655

ABSTRACT

As an important model industrial microorganism, Escherichia coli has been widely used in pharmaceutical, chemical industry and agriculture. In the past 30 years, a variety of new strategies and techniques, including artificial intelligence, gene editing, metabolic pathway assembly, and dynamic regulation have been used to design, construct, and optimize E. coli cell factories, which remarkably improved the efficiency for biotechnological production of chemicals. In this review, three key aspects for constructing E. coli cell factories, including pathway design, pathway assembly and regulation, and optimization of global cellular performance, are summarized. The technologies that have played important roles in metabolic engineering of E. coli, as well as their future applications, are discussed.


Subject(s)
Artificial Intelligence , Escherichia coli/genetics , Gene Editing , Metabolic Engineering , Metabolic Networks and Pathways/genetics
16.
Chinese Journal of Biotechnology ; (12): 1494-1509, 2021.
Article in Chinese | WPRIM | ID: wpr-878651

ABSTRACT

In 1990s, Bailey and Stephanopoulos put forward the concept of classic metabolic engineering, aiming to use DNA recombination technology to rewire metabolic network to achieve improved cell performance and increased target products. In the last 30 years since the birth of metabolic engineering, life science have flourished, and new disciplines such as genomics, systems biology and synthetic biology have emerged, injecting new connotations and vitality into the development of metabolic engineering. Classic metabolic engineering research has entered into an unprecedented stage of systems metabolic engineering. The application of synthetic biology tools and strategies, such as omics technology, genomic-scale metabolic model, parts assembly, circuits design, dynamic control, genome editing and many others, have greatly improved the design, build, and rewiring capabilities of complex metabolism. The intervention of machine learning and the combination of evolutionary engineering and metabolic engineering will further promote the development of systems metabolic engineering. This paper analyzes the development of metabolic engineering in the past 30 years and summarizes the novel theories, techniques, strategies, and applications of metabolic engineering that have emerged over the past 30 years.


Subject(s)
Gene Editing , Metabolic Engineering , Metabolic Networks and Pathways/genetics , Synthetic Biology , Systems Biology
17.
Chinese Journal of Biotechnology ; (12): 1385-1395, 2021.
Article in Chinese | WPRIM | ID: wpr-878640

ABSTRACT

Streptococcus pyogenes Cas9 (SpCas9) has become a powerful genome editing tool, but has a limited range of recognizable protospacer adjacent motifs (PAMs) and shows off-target effects. To address these issues, we present a rational approach to optimize the xCas9 mutant derived from SpCas9 by directed evolution. Firstly, energy minimization with the Rosetta program was applied to optimize the three-dimensional structure of Cas9 to obtain the lowest energy conformation. Subsequently, combinatorial mutations were designed based on the mutations sites of xCas9 acquired during the directed evolution. Finally, optimal mutants were selected from the designed mutants by free energy ranking and subjected to experimental verification. A new mutant yCas9 (262A/324R/409N/480K/543D/694L/1219T) with multiple PAM recognition ability and low off-target effects was obtained and verified by DNA cleavage experiments. This mutant recognizes the NG, GAA and GAT PAMs and shows low off-target DNA cleavage activity guided by mismatched sgRNA, thus provides a gene editing tool with potential applications in biomedical field. Furthermore, we performed molecular dynamics simulations on the structures of SpCas9, xCas9 and yCas9 to reveal the mechanisms of their PAM recognition and off-target effects. These may provide theoretical guidance for further optimization and modification of CRISPR/Cas9 proteins.


Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Editing , RNA, Guide/genetics , Streptococcus pyogenes/metabolism
18.
Chinese Journal of Biotechnology ; (12): 1205-1228, 2021.
Article in Chinese | WPRIM | ID: wpr-878625

ABSTRACT

Genome editing is a genetic manipulation technique that can modify DNA sequences at the genome level, including insertion, knockout, replacement and point mutation of specific DNA fragments. The ultimate principle of genome editing technology relying on engineered nucleases is to generate double-stranded DNA breaks at specific locations in genome and then repair them through non-homologous end joining or homologous recombination. With the intensive study of these nucleases, genome editing technology develops rapidly. The most used nucleases include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats associated Cas proteins. Based on introducing the development and principles of above mentioned genome editing technologies, we review the research progress of CRISPR/Cas9 system in the application fields of identification of gene function, establishment of disease model, gene therapy, immunotherapy and its prospect.


Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing , Technology , Transcription Activator-Like Effector Nucleases/metabolism
19.
Chinese Journal of Biotechnology ; (12): 980-990, 2021.
Article in Chinese | WPRIM | ID: wpr-878608

ABSTRACT

Aspergillus niger is a vital industrial workhouse widely used for the production of organic acids and industrial enzymes. This fungus is a crucial cell factory due to its innate tolerance to a diverse range of abiotic conditions, high production titres, robust growth during industrial scale fermentation, and status as a generally recognized as safe (GRAS) organism. Rapid development of synthetic biology and systems biology not only offer powerful approaches to unveil the molecular mechanisms of A. niger productivity, but also provide more new strategies to construct and optimize the A. niger cell factory. As a new generation of genome editing technology, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) system brings a revolutionary breakthrough in targeted genome modification for A. niger. In this review, we focus on current advances to the CRISPR/Cas genome editing toolbox, its application on gene modification and gene expression regulation in this fungal. Moreover, the future directions of CRISPR/Cas genome editing in A. niger are highlighted.


Subject(s)
Aspergillus niger/genetics , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing , Genome
20.
Chinese Journal of Biotechnology ; (12): 950-965, 2021.
Article in Chinese | WPRIM | ID: wpr-878606

ABSTRACT

Saccharomyces cerevisiae is one of the most important hosts in metabolic engineering. Advanced gene editing technology has been widely used in the design and construction of S. cerevisiae cell factories. With the rapid development of gene editing technology, early gene editing technologies based on recombinase and homologous recombination have been gradually replaced by new editing systems. In this review, the principle and application of gene editing technology in S. cerevisiae are summarized. Here, we first briefly describe the classical gene editing techniques of S. cerevisiae. Then elaborate the genome editing system of MegNs, ZFNs and TALENs based on endonuclease. The latest research progress is especially introduced and discussed, including the CRISPR/Cas system, multi-copy integration of heterologous metabolic pathways, and genome-scale gene editing. Finally, we envisage the application prospects and development directions of Saccharomyces cerevisiae gene editing technology.


Subject(s)
CRISPR-Cas Systems/genetics , Endonucleases/genetics , Gene Editing , Saccharomyces cerevisiae/genetics , Technology
SELECTION OF CITATIONS
SEARCH DETAIL