ABSTRACT
The CRISPR-Cas9 system is composed of a clustered regularly interspaced short palindromic repeat (CRISPR) and its associated proteins, which are widely present in bacteria and archaea, serving as a specific immune protection against viral and phage secondary infections. CRISPR-Cas9 technology is the third generation of targeted genome editing technologies following zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). The CRISPR-Cas9 technology is now widely used in various fields. Firstly, this article introduces the generation, working mechanism and advantages of CRISPR-Cas9 technology; secondly, it reviews the applications of CRISPR-Cas9 technology in gene knockout, gene knock-in, gene regulation and genome in breeding and domestication of important food crops such as rice, wheat, maize, soybean and potato. Finally, the article summarizes the current problems and challenges encountered by CRISPR-Cas9 technology and prospects future development and application of CRISPR-Cas9 technology.
Subject(s)
Gene Editing , CRISPR-Cas Systems/genetics , Plant Breeding , Crops, Agricultural/genetics , TechnologyABSTRACT
Cervical cancer is the fourth-ranked malignant tumor of female cancer in the world, and it seriously threatens women’s health. The main treatment options for patients with cervical cancer are surgery or concurrent chemoradiotherapy. With the development of medical research, researchers are committed to exploring more effective and specific treatment options in order to increase the treatment options for cervical cancer and improve the treatment effect. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a method in which the Cas9 protein uses guide RNA (gRNA) to target the target gene and achieve precise editing of the target gene. At present, CRISPR/Cas9 technology has become a promising and powerful gene editing tool, a new and effective targeted therapy that has been applied in the treatment of various tumors. The research progress of CRISPR/Cas9 technology in the treatment of cervical cancer is mainly reviewed in terms of action targets, combination therapy strategies, and related drug resistance gene screening in order to provide new strategies for the treatment of cervical cancer.
ABSTRACT
OsRhoGDI2 was isolated as a putative partner of Rho protein family member OsRacD from rice panicles by yeast two-hybrid, but its function remains unknown. In order to identify the function of OsRhoGDI2, OsRhoGDI2 knockout mutants were created by CRISPR/Cas9 technology. The results showed that two different homozygous mutants were obtained in T0 generation, and eight kinds homozygous mutants were identified in T1 generation. Sequence analysis revealed that the base substitution or base deletion occurred near the editing targets of the gene in knockout rice, and it could be expected that the truncated OsRhoGDI2 proteins lacking the RhoGDI conserved domain would be generated. Phenotype analysis showed that the OsRhoGDI2 knockout rice plants were significantly lower than the control plants. Statistical analysis confirmed that the significant decrease of plant height was due to the shortening of the second and third internodes, suggesting that OsRhoGDI2 gene may be related with rice height control.
Subject(s)
CRISPR-Cas Systems , Genes, Plant , Genetics , Oryza , Genetics , Plants, Genetically Modified , rho Guanine Nucleotide Dissociation Inhibitor beta , GeneticsABSTRACT
Objective · To establish the Fbxo22 knockout mouse model and study the biological function of FBXO22. Methods · The Fbxo22 knockout mice were generated by CRISPR-Cas9 technology. The number, appearance, weight of different embryos and mice were measured. Meanwhile, the food intake and survival of Fbxo22-/- mice were analyzed. Results · Although the Fbxo22-/- embryos were present at approximately Mendelian ratios on embryonic day 17.5/18.5, most of them died within 48 hours of birth. Furthermore, those surviving Fbxo22-/- mice showed reduced body size and food intake and decreased life span. Conclusion · FBXO22 is an important, albeit not essential, protein for early postnatal survival and normal development.
ABSTRACT
Objective • To establish the Fbxo22 knockout mouse model and study the biological function of FBXO22. Methods • The Fbxo22 knockout mice were generated by CRISPR-Cas9 technology. The number, appearance, weight of different embryos and mice were measured. Meanwhile, the food intake and survival of Fbxo22-/- mice were analyzed. Results • Although the Fbxo22-/- embryos were present at approximately Mendelian ratios on embryonic day 17.5/18.5, most of them died within 48 hours of birth. Furthermore, those surviving Fbxo22-/- mice showed reduced body size and food intake and decreased life span. Conclusion • FBXO22 is an important, albeit not essential, protein for early postnatal survival and normal development.
ABSTRACT
Objective To construct ARID2 knockout human liver cancer cell line Hep3B by CRISPR/Cas9 system, explore the effect of ARID2 knockout on the proliferation of Hep3B, and the differences in gene expression between wild type and ARID2 knockout Hep3B cell lines. Methods The plasmid lentiCRISPRv2 was constructed with ARID2 knockout plasmid, and then transfected Hep3B cells; The positive cells were selected by puromycin, sorted by flow cytometry and cultured to obtain the monoclonal cell lines; ARID2 knockout Hep3B cell lines were identified by Western blotting and Sanger sequencing; The effect of ARID2 knockout on the proliferation of Hep3B cell line was detected by CCK-8 method; RNA-seq was used to analyze the differentially expressed genes between wild type Hep3B cells and ARID2 knockout Hep3B cells, and the results of RNA-seq were validated by Real-time quantitative PCR (RT-qPCR). The possible biology functions of ARID2 were explored through Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG Pathway), GO Biological Processes and Gene set enrichment analysis (GSEA). Results Two ARID2 knockout Hep3B cell lines were successfully constructed. Compared with the wild type cell line, the proliferation of ARID2 knockout cell lines was significantly accelerated (P<0.05). A total of 85 differentially expressed genes were identified by RNA-seq analysis between ARID2 knockout cell lines and wild type cell line, of which 17 genes were up-regulated and 68 down-regulated. The mRNA expression of 10 differentially expressed genes were validated by RT-qPCR, the verification results were consistent with that by RNA-seq. Results of KEGG Pathway, GO Biological Processes and GSEA indicated that ARID2 genes might be involved in such biological processes as protein processing and transport, chemokine signaling pathway, Wnt signaling pathway, complement and coagulation cascade reaction, epithelial-mesenchymal transition (EMT), glycolysis, TGF-β signaling pathway, and TNF-α/NF-KB signaling pathway, et al. Conclusion ARID2 knockout can promote proliferation of Hep3B cell line; and ARID2 may play an important role by variety of biological processes in tumor proliferation, invasion, metastasis and tumor microenvironment.
ABSTRACT
The clustered regulatory interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system is the part of the prokaryotic immune system, which could recognize and delete the exogenous sequences originated from virus or plasmid. Based on its mechanism, CRISPR-Cas9 system was developed into the new generation of gene editing tool. Compared to the existed technologies such as ES targeting, ZFN or TALEN, CRISPR-Cas9 system is a more efficient, economical and promising approach to manipulate the genome. In this review, we summarize the research progress about CRISPR-Cas9 technology, especially the latest applications in gene therapy studies of human diseases.