During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.
Humans ; Animals ; Gene Editing ; CRISPR-Cas Systems/genetics* ; HEK293 Cells ; Homologous Recombination ; DNA

Gene editing technology can be used to modify the genome of Escherichia coli for the investigation of gene functions, or to change the metabolic pathways for the efficient production of high-value products in engineered strains with genetic stability. A variety of gene editing technologies have been applied in prokaryotes, such as λ-Red homologous recombination and CRISPR/Cas9. As a traditional gene editing technique, λ-Red recombination is widely used. However, it has a few shortcomings, such as the limited integration efficiency by the integrated fragment size, the cumbersome gene editing process, and the FRT scar in the genome after recombination. CRISPR/Cas9 is widely used for genome editing at specific sites, which requires specific DNA segments according to the editing site. As the understanding of the two technologies deepens, a variety of composite gene editing techniques have been developed, such as the application of λ-Red homologous recombination in combination with homing endonucleaseⅠ-SceⅠ or CRISPR/Cas9. In this review, we summarized the basic principles of common gene editing techniques and composite gene editing techniques, as well as their applications in Escherichia coli, which can provide a basis for the selection of gene editing methods in prokaryotes.
CRISPR-Cas Systems/genetics* ; Escherichia coli/genetics* ; Gene Editing ; Homologous Recombination ; Technology

The establishment and development of gene knockout mice have provided powerful support for the study of gene function and the treatment of human diseases. Gene targeting and gene trap are two techniques for generating gene knockout mice from embryonic stem cells. Gene targeting replaces endogenous knockout gene by homologous recombination. There are two ways to knock out target genes: promoter trap and polyA trap. In recent years, many new gene knockout techniques have been developed, including Cre/loxP system, CRISP/Cas9 system, latest ZFN technology and TALEN technology. This article focuses on the several new knockout mouse techniques.
Animals ; Disease Models, Animal ; Embryonic Stem Cells ; Gene Knockout Techniques ; trends ; Gene Targeting ; trends ; Homologous Recombination ; Humans ; Mice ; Mice, Knockout

Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.
CRISPR-Cas Systems ; Gene Editing ; Genetic Engineering ; Homologous Recombination

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. RAG-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of RAG-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of RAG-2, we recently established a new RAG-2 knockout FVB mouse line (RAG-2(−/−)) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. RAG-2(−/−) mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in RAG-2(−/−) mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in RAG-2(−/−) mice. These findings indicate that our RAG-2(−/−) mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.
Animals ; Atrophy ; B-Lymphocytes ; Bone Marrow ; Genome ; Homologous Recombination ; Humans ; Immunoglobulins ; Killer Cells, Natural ; Lymph Nodes ; Lymphocytes ; Lymphopenia* ; Megakaryocytes ; Methods ; Mice* ; Mice, Knockout ; Negotiating ; Phenotype ; Receptors, Antigen, T-Cell ; Recombination, Genetic ; Sensitivity and Specificity ; Severe Combined Immunodeficiency ; Spleen ; T-Lymphocytes ; Thymus Gland

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p < 0.01) in chickens inoculated with C79-13ΔcrpΔasd (pYA-HN) than with C79-13ΔcrpΔasd (pYA) but were not significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.
Animals ; Antibodies ; Bacteria ; Chickens ; Hemagglutination ; HN Protein ; Homologous Recombination ; Immunoglobulin A, Secretory ; Immunoglobulin G ; Lymphocytes ; Methods ; Newcastle disease virus ; Newcastle Disease ; Plasmids ; Poultry ; Salmonella ; Suicide ; Vaccines

OBJECTIVE: To define genetic profiling of homologous recombination (HR) deficiency in Chinese ovarian cancer patients. METHODS: we have applied next-generation sequencing to detect deleterious mutations through all exons in 31 core HR genes. Paired whole blood and frozen tumor samples from 50 Chinese women diagnosed with epithelial ovarian carcinomas were tested to identify both germline and somatic variants. RESULTS: Deleterious germline HR-mutations were identified in 36% of the ovarian cancer patients. Another 5 patients had only somatic mutations. BRCA2 was most frequently mutated. Three out of the 5 somatic mutations were in RAD genes and a wider distribution of other HR genes was involved in non-serous carcinomas. BRCA1/2-mutation carriers had favorable platinum sensitivity (relative risk, 1.57, p<0.05), resulting in a 100% remission probability and survival rate. In contrast, mutations in other HR genes predicted poor prognosis. However, multivariate analysis demonstrated that platinum sensitivity and optimal cytoreduction were the independent impact factors influencing survival (hazards ratio, 0.053) and relapse (hazards ratio, 0.247), respectively. CONCLUSION: our results suggest that a more comprehensive profiling of HR defect than merely BRCA1/2 could help elucidate tumor heterogeneity and lead to better stratification of ovarian cancer patients for individualized clinical management.
Asian Continental Ancestry Group* ; Exons ; Female ; Homologous Recombination* ; Humans ; Multivariate Analysis ; Ovarian Neoplasms* ; Platinum ; Population Characteristics ; Prognosis ; Recurrence ; Survival Rate

Pluripotent stem cells (PSCs) are a useful source of cells for exploring the role of genes related with early developmental processes and specific diseases due to their ability to differentiate into all somatic cell types. Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system has proven to be a robust tool for targeted genetic modification. Here, we generated miR-451-deficient PSCs using the CRISPR/Cas9 system with PCR-based homologous recombination donor and investigated the impact of its deletion on self-renewal and hematopoietic development. CRISPR/Cas9-mediated miR-451 knockout did not alter the gene expressions of pluripotency, cellular morphology, and cell cycle, but led to impaired erythrocyte development. These findings propose that a combination of PSCs and CRISPR/Cas9 system could be useful to promote biomedical applications of PSCs by elucidating the function and manipulating of specific miRNAs during lineage specification and commitment.
Animals ; Cell Cycle ; Clustered Regularly Interspaced Short Palindromic Repeats* ; Erythrocytes ; Gene Expression ; Hematopoiesis ; Homologous Recombination ; Humans ; Mice* ; MicroRNAs ; Mouse Embryonic Stem Cells* ; Pluripotent Stem Cells ; Tissue Donors

Breast carcinoma is a heterogeneous disease that has exhibited rapid resistance to treatment in the last decade. Depending genotype and phenotype of breast cancer, there are discernible differences in DNA repair protein responses including DNA double strand break repair. It is a fact that different molecular sub-types of breast carcinoma activate these dedicated protein pathways in a distinct manner. The DNA double-strand damage repair machinery is manipulated by breast carcinoma to selectively repair the damage or insults inflicted by the genotoxic effects of chemotherapy or radiation therapy. The two DNA double-strand break repair pathways employed by breast carcinoma are homologous recombination and non-homologous end joining. In recent decades, therapeutic interventions targeting one or more factors involved in repairing DNA double-strand breaks inflicted by chemo/radiation therapy have been widely studied. Herein, this review paper summarizes the recent evidence and ongoing clinical trials citing potential therapeutic combinatorial interventions targeting DNA double-strand break repair pathways in breast carcinoma.
Breast Neoplasms* ; Breast* ; DNA Repair ; DNA* ; Drug Therapy ; Genotype ; Homologous Recombination ; Phenotype ; Radiotherapy

PURPOSE: Heterochromatin protein 1gamma (HP1gamma) interacts with chromosomes by binding to lysine 9-methylated histone H3 or DNA/RNA. HP1gamma is involved in various biological processes. The purpose of this study is to gain an understanding of how HP1gamma functions in these processes by identifying HP1gamma-binding proteins using mass spectrometry. MATERIALS AND METHODS: We performed affinity purification of HP1gamma-binding proteins using G1/S phase or prometaphase HEK293T cell lysates that transiently express mock or FLAG-HP1gamma. Coomassie staining was performed for HP1gamma-binding complexes, using cell lysates prepared by affinity chromatography FLAG-agarose beads, and the bands were digested and then analyzed using a mass spectrometry. RESULTS: We identified 99 HP1gamma-binding proteins with diverse cellular functions, including spliceosome, regulation of the actin cytoskeleton, tight junction, pathogenic Escherichia coli infection, mammalian target of rapamycin signaling pathway, nucleotide excision repair, DNA replication, homologous recombination, and mismatch repair. CONCLUSION: Our results suggested that HP1gamma is functionally active in DNA damage response via protein-protein interaction.
Actin Cytoskeleton ; Biological Processes ; Chromatography, Affinity ; DNA Damage* ; DNA Mismatch Repair ; DNA Repair ; DNA Replication ; DNA* ; Escherichia coli Infections ; Heterochromatin* ; Histones ; Homologous Recombination ; Lysine ; Mass Spectrometry ; Prometaphase ; Sirolimus ; Spliceosomes ; Tight Junctions