Biomedical Research ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans

Central to the core principle of cell theory, depicting cells' history, state and fate is a fundamental goal in modern biology. By leveraging clonal analysis and single-cell RNA-seq technologies, single-cell lineage tracing provides new opportunities to interrogate both cell states and lineage histories. During the past few years, many strategies to achieve lineage tracing at single-cell resolution have been developed, and three of them (integration barcodes, polylox barcodes, and CRISPR barcodes) are noteworthy as they are amenable in experimentally tractable systems. Although the above strategies have been demonstrated in animal development and stem cell research, much care and effort are still required to implement these methods. Here we review the development of single-cell lineage tracing, major characteristics of the cell barcoding strategies, applications, as well as technical considerations and limitations, providing a guide to choose or improve the single-cell barcoding lineage tracing.
Animals ; Cell Lineage/genetics* ; Clustered Regularly Interspaced Short Palindromic Repeats

The CRISPR-Cas9 system is a new targeted nuclease for genome editing, which can directly introduce modifications at the targeted genomic locus. The system utilizes a short single guide RNA (sgRNA) to direct the endonuclease Cas9 in the genome. Upon targeting, Cas9 can generate DNA double-strand breaks (DSBs). As such DSBs are repaired by non-homologous end joining (NHEJ) or homology directed repair (HDR), therefore facilitates introduction of random or specific mutations, repair of endogenous mutations, or insertion of DNA elements. The system has been successfully used to generate gene targeted cell lines including those of human, animals and plants. This article reviews recent advances made in this rapidly evolving technique for the generation of animal models for human diseases.
Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; genetics ; Disease Models, Animal ; Humans ; RNA Editing ; genetics

This study was aimed to explore the features of clustered regularly interspaced short palindromic repeats (CRISPR) structures in Shigella by using bioinformatics. We used bioinformatics methods, including BLAST, alignment and RNA structure prediction, to analyze the CRISPR structures of Shigella genomes. The results showed that the CRISPRs existed in the four groups of Shigella, and the flanking sequences of upstream CRISPRs could be classified into the same group with those of the downstream. We also found some relatively conserved palindromic motifs in the leader sequences. Repeat sequences had the same group with corresponding flanking sequences, and could be classified into two different types by their RNA secondary structures, which contain "stem" and "ring". Some spacers were found to homologize with part sequences of plasmids or phages. The study indicated that there were correlations between repeat sequences and flanking sequences, and the repeats might act as a kind of recognition mechanism to mediate the interaction between foreign genetic elements and Cas proteins.
Base Sequence ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology ; Genome, Bacterial ; Plasmids ; Shigella ; genetics

Genome editing refers to the experimental methods to targeted modify specific loci in the genomic DNA sequence. In recent years, engineered endonucleases, including ZFN, TALEN and CRISPR/Cas, have been developed as a new-generation genome editing technique, and greatly improved the feasibility of gene function analyses, gene therapy, etc. Here, we briefly summarize the basic principle, developmental process and applications of this technology.
Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases ; genetics ; Genetic Engineering ; methods ; Genetic Therapy ; Genome ; Genomics

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.
CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Genetic Therapy ; Humans ; Plasmids

Gene editing is a technique for modifying gene fragments. The novel gene editing technology focuses on the field of artificial nuclease cleavage technology, mainly ZFN technology, TALEN technology, CRISPR technology and base editing technology. The continuous improvement of gene editing technology has promoted the rapid development of agriculture, animal husbandry and biomedicine, but at the same time, technical defects and ethical controversy have brought enormous challenges to its own development. This article will briefly discuss the development and challenges of gene editing technology, as well as the views at home and abroad, and hope to inspire readers to recognize gene editing technology.
Agriculture ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; Endonucleases ; Gene Editing

The CRISPR/Cas9 system, consisting of Cas9 nuclease and single guide RNA (sgRNA), is an emerging gene editing technology that can perform gene reprogramming operations such as deletion, insertion, and point mutation on DNA sequences targeted by sgRNA. In addition, CRISPR/dCas9 (a mutant that loses Cas9 nuclease activity) still retains the ability of sgRNA to target DNA. The fusion of dCas9 protein with transcriptional activator (CRISPRa) can activate the expression of the target gene, and fusion transcriptional repressors (CRISPRi) can also be used to suppress target gene expression. Efficient delivery of the CRISPR/Cas9 system is one of the main problems limiting its wide clinical application. Viral vectors are widely used to efficiently deliver CRISPR/Cas9 elements, but non-viral vector research is more attractive in terms of safety, simplicity, and flexibility. In this review, we summarize the principles and research advances of CRISPR technology, including CRISPR/ Cas9 delivery vectors, delivery methods, and obstacles to the delivery, and review the progress of CRISPR-based research in bone and cartilage tissue engineering. Finally, the challenges and future applications of CRISPR technology in bone and cartilage tissue engineering are discussed.
CRISPR-Cas Systems ; Cartilage ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide ; Tissue Engineering

Recently, with the development and the continuous improvement of various CRISPR systems represented by CRISPR/Cas9, gene editing technology has been gradually improved, and widely applied to the preparation of animal models of human diseases. The gene edited animal models provide important materials for the study of pathogenesis, pathological process, prevention and treatment of human diseases. At present, the gene edited animal models used in human disease research include mainly the rodent models represented by mice and rats, and large animal models represented by pigs. Among them, rodents differ greatly from humans in all aspects of their bodies and have short life span as well, which cannot provide effective evaluation and long-term tracking for the research and treatment of human diseases. On the other hand, pig is closer to human in physiology, anatomy, nutrition and genetics, which provides an important animal model in the field of organ transplantation and human disease research. In this paper, the application of the gene edited animal models was summarized in the researches of 5 human diseases such as neurodegenerative diseases, familial hypertrophic cardiomyopathy, cancer, immunodeficiency diseases and metabolic diseases. We hope this paper will provide a reference for the research of human diseases and the preparation of relative animal models.
Animals ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Models, Animal ; Gene Editing ; Humans

Gluconacetobacter xylinus is a primary strain producing bacterial cellulose (BC). In G. xylinus, BcsD is a subunit of cellulose synthase and is participated in the assembly process of BC. A series of G. xylinus with different expression levels of the bcsD gene were obtained by using the CRISPR/dCas9 technique. Analysis of the structural characteristics of BC showed that the crystallinity and porosity of BC changed with the expression of bcsD. The porosity varied from 59.95%-84.05%, and the crystallinity varied from 74.26%-93.75%, while the yield of BC did not decrease significantly upon changing the expression levels of bcsD. The results showed that the porosity of bacterial cellulose significantly increased, while the crystallinity was positively correlated with the expression of bcsD, when the expression level of bcsD was below 55.34%. By altering the expression level of the bcsD gene, obtaining BC with different structures but stable yield through a one-step fermentation of G. xylinus was achieved.
Cellulose/chemistry* ; Clustered Regularly Interspaced Short Palindromic Repeats ; Fermentation ; Gluconacetobacter xylinus/metabolism*