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

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

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

OBJECTIVETo detect the molecular characteristics of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) in Shigella and to analyze the distribution of CRISPR related to the time of isolation.

METHODSOf the 52 Shigella strains, 41 were isolated from Henan, 6 from Jiangxi and 5 isolated from Beijing. Both CRISPR locus of S1, S2, S3 and S4 in Shigella were detected by polymerase chain reaction (PCR). The PCR products were sequenced and compared.

RESULTSThe positive rates of CRISPR locus in Shigella were 33.69% (S1), 50.00% (S2), 82.69% (S3) and 73.08% (S4), respectively. Two subtypes were discovered in S1 and S3 locus. Three subtypes were discovered in S2 locus. Four different subtypes were discovered in S4 locus. The isolates from Henan strains were divided into two groups by the time of isolation. Distributions of S1 were different, before or after 2004, on Shigella. S1 could not be detected after 2004. There were no statistical differences of S2, S3 and S4 in two groups.

CONCLUSIONDifferent CRISPR subtypes or Shigella were discovered. A significant correlation was noticed between the CRISPR S1 related to the time of isolation but not between S2, S3 or S4 on the time of isolation.

As the breakthrough in gene editing, represented by CRISPR/Cas9, gene manipulations now are more maneuverable, economically feasible and time saving. It is possible for China to catch an overtaking in researching and industrializing of downside sections (especially the application of plant gene editing), also the incubation of professional companies in gene editing fields. For this consideration, it is necessary and urgent to find the key demands and potential application for gene editing in China. Questionnaire and statistic analysis were carried out to find the key demands and the most potential application fields of the development for gene editing. Firstly, an ordered multi-classification Logistic regression model was established following with dependent variable analysis. Eight out of 24 questionnaires questions in 4 categories were regarded as independent variables with significance test. Then, regression model based on ordered multi-classification logistic method was established to analyze the specific impact of different options on the development of gene editing. The results showed that most researchers in the field of gene editing take the view that development of potential competitive advantages lies in the field of plant science. The results also showed that major gene editing experts believe more attention should be paid on how to carry out technology industrialization while focusing on basic technology development, as well as the development of potential competitive advantages of gene editing technology in plant field. To promote the development of gene editing in China, not only the participation of scientific research institution was needed, but also the synergy of various forces both universities and governments. It is urgent both properly guiding public opinion on gene editing and establishing a national safety standard system. At the same time, the key point of technology risk avoidance should be put on biological weapons and bioterrorism, gene editing related infectious disease, and the potential risk of species genetic change on the ecological environment, etc.
CRISPR-Cas Systems/genetics* ; China ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Plants/genetics*

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