ABSTRACT
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.
Subject(s)
Humans , Animals , Gene Editing , CRISPR-Cas Systems/genetics , HEK293 Cells , Homologous Recombination , DNAABSTRACT
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.
Subject(s)
Animals , Humans , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Disease Models, Animal , Gene EditingABSTRACT
In order to study the modulatory effects of compound polysaccharide Erweikang on the immune function of the immunosuppressive mice, the positive immune modulation of Erweikang at different doses and different time points was compared when normal saline and cyclophosphamide served as controls. The results showed that different doses of Erweikang could effectively reverse the decreased induction levels of NK, IL-2, gamma-IFN and the contents of TNF and IgF of the immunosuppressive mice after administration of Erweikang for 7 to 14 days. It was suggested that compound polysaccharide Erweikang presents positive immune modulation in dose- and time-dependent manners.