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.
CRISPR-Cas Systems/genetics* ; Chromatin ; Endonucleases ; Gene Editing

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.
CRISPR-Cas Systems/genetics* ; Endonucleases/genetics* ; Gene Editing ; Saccharomyces cerevisiae/genetics* ; Technology

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.
Animals ; Bombyx/metabolism* ; CRISPR-Cas Systems/genetics* ; Endonucleases/genetics* ; Gene Editing ; RNA, Guide/genetics*

BACKGROUND: Transcription factor (TF) can bind specific sequences that either promotes or represses the transcription of target genes, and exerts important effects on tumorigenesis, migration, invasion. Staphylococcal nuclease-containing structural domain 1 (SND1), which is a transcriptional co-activator, is considered as a promising target for tumor therapy. However, its role in lung adenocarcinoma (LUAD) remains unclear. This study aims to explore the role of SND1 in LUAD. METHODS: Data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and Human Protein Atlas (HPA) database was obtained to explore the association between SND1 and the prognosis, as well as the immune cell infiltration, and subcellular localization in LUAD tissues. Furthermore, the functional role of SND1 in LUAD was verified in vitro. EdU assay, CCK-8 assay, flow cytometry, scratch assay, Transwell assay and Western blot were performed. RESULTS: SND1 was found to be upregulated and high expression of SND1 is correlated with poor prognosis of LUAD patients. In addition, SND1 was predominantly present in the cytoplasm of LUAD cells. Enrichment analysis showed that SND1 was closely associated with the cell cycle, as well as DNA replication, and chromosome segregation. Immune infiltration analysis showed that SND1 was closely associated with various immune cell populations, including T cells, B cells, cytotoxic cells and dendritic cells. In vitro studies demonstrated that silencing of SND1 inhibited cell proliferation, invasion and migration of LUAD cells. Besides, cell cycle was blocked at G1 phase by down-regulating SND1. CONCLUSIONS SND1 might be an important prognostic biomarker of LUAD and may promote LUAD cells proliferation and migration.
Humans ; Prognosis ; Proteomics ; Lung Neoplasms/genetics* ; Oncogenes ; Adenocarcinoma of Lung/genetics* ; Biomarkers ; Endonucleases/genetics*

Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.
Chromosomes, Bacterial ; genetics ; DNA ; Endonucleases ; metabolism ; Escherichia coli ; genetics ; Genetic Engineering ; methods ; Recombination, Genetic ; Sequence Deletion

Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.
Animals ; CRISPR-Cas Systems/genetics* ; DNA/genetics* ; Embryo, Mammalian/metabolism* ; Endonucleases/metabolism* ; Gene Editing ; Mammals/metabolism*

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

OBJECTIVETo construct a system of I-SceI and induce a site-specific DNA double-strand break (DSB) in the genome of HepG2 for using this system in future exploration of the potential mechanisms of HBV integration by DSB repair.

METHODSThe eukaryotic expression plasmid pEGFP2 was constructed and transfected into human hepatoma cell line HepG2. The positive neomycin-resistant transfected cell clones were generated by G418 selection. Then the positive cells containing an 18-bp I-SceI endonuclease site were transfected transiently with pCMV(3NLS) I-SceI, an I-SceI expression plasmid. At 24 h post-transfection with pCMV (3NLS) I-SceI, gamma-H2AX, as an early cellular marker of DSB, was detected using immunocytochemistry and Western blot analysis.

RESULTSRestriction analysis and DNA sequencing verified that the plasmid pEGFP2 was successfully constructed. gamma-H2AX increased significantly in cells transfected with the I-SceI system.

CONCLUSIONSGenomic DSB can be induced into HepG2 by introducing an I-SceI system. The cell model could provide us with a practical tool for further study to see if DSB is a potential target for HBV integration.

Carcinoma, Hepatocellular ; genetics ; DNA Breaks, Double-Stranded ; DNA Repair ; Flap Endonucleases ; genetics ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; Plasmids

BACKGROUNDSeveral studies have evaluated the association between polymorphisms of encoding excision repair cross complementation group 1 (ERCC1) enzyme and lung cancer risk in diverse populations but with conflicting results. By pooling the relatively small samples in each study, it is possible to perform a meta-analysis of the evidence by rigorous methods.

METHODSEmbase, Ovid, Medline and Chinese National Knowledge Infrastructure were searched. Additional studies were identified from references in original studies or review articles. Articles meeting the inclusion criteria were reviewed systematically, and the reported data were aggregated using the statistical techniques of meta-analysis.

RESULTSWe found 3810 cases with lung cancer and 4332 controls from seven eligible studies. T19007C polymorphism showed no significant effect on lung cancer risk (C allele vs. T allele: odds ratio (OR) = 0.91, 95% confidence interval (CI) = 0.80 - 1.04; CC vs. TT: OR = 0.76, 95%CI = 0.56 - 1.02; CC vs. (CT + TT): OR = 0.96, 95%CI = 0.84 - 1.10). Similarly, there was no significant main effects for T19007C polymorphism on lung cancer risk when stratified analyses by ethnicity (Chinese or Caucasian). No significant association was found between C8092A polymorphism (3060 patients and 2729 controls) and the risk of lung cancer (A allele vs. C allele: OR = 1.03, 95%CI = 0.95 - 1.11; AA vs. CC: OR = 1.08, 95%CI = 0.88 - 1.33; AA vs. (AC + CC): OR = 1.08, 95%CI = 0.88 - 1.31).

CONCLUSIONWe found little evidence of an association between the T1900C or C8092A polymorphisms of ERCC 1 and the risk of lung cancer in Caucasian or Han Chinese people.

Asian Continental Ancestry Group ; genetics ; DNA-Binding Proteins ; genetics ; Endonucleases ; genetics ; Genetic Predisposition to Disease ; genetics ; Humans ; Lung Neoplasms ; genetics ; Polymorphism, Genetic ; genetics

BACKGROUND: The pathogenesis of osteosarcoma (OS) is still unclear, and it is still necessary to find new targets and drugs for anti-OS. This study aimed to investigate the role and mechanism of the anti-OS effects of miR-296-5p. METHODS: We measured the expression of miR-296-5p in human OS cell lines and tissues. The effect of miR-296-5p and its target gene staphylococcal nuclease and tudor domain containing 1 on proliferation, migration, and invasion of human OS lines was examined. The Student's t test was used for statistical analysis. RESULTS: We found that microRNA (miR)-296-5p was significantly downregulated in OS cell lines and tissues (control vs. OS, 1.802 ± 0.313 vs. 0.618 ± 0.235, t = 6.402, P < 0.01). Overexpression of miR-296-5p suppressed proliferation, migration, and invasion of OA cells. SND1 was identified as a target of miR-296-5p by bioinformatic analysis and dual-luciferase reporter assay. Overexpression of SND1 abrogated the effects induced by miR-296-5p upregulation (miRNA-296-5p vs. miRNA-296-5p + SND1, 0.294 ± 0.159 vs. 2.300 ± 0.277, t = 12.68, P = 0.003). CONCLUSION Our study indicates that miR-296-5p may function as a tumor suppressor by targeting SND1 in OS.
Bone Neoplasms/genetics* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Endonucleases/genetics* ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Humans ; MicroRNAs/genetics* ; Osteosarcoma/genetics*