crisprcas9-ribonucleoprotein-mediated-precise-gene-editing-tube

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

University of Michigan Medical Center

CETP (cholesteryl ester transfer protein) plays an important role in lipoprotein metabolism; however, whether inhibition of CETP activity can prevent cardiovascular disease remains controversial.

Deficiency of Cholesteryl Ester Transfer Protein Protects Against Atherosclerosis in Rabbits.

Immunodeficient mice have been used predominantly in biomedical research. Realizing that large animal species may have an enhanced ability to predict clinical outcome relative to mice, we worked to develop immunodeficient rabbits by CRISPR/Cas9. We first demonstrated that multiplex embryo transfer efficiently produced multiple lines of single-gene mutant (SGM) founders. Embryos microinjected with single sgRNA targeting FOXN1, RAG2, IL2RG or PRKDC were pooled for embryo transfer. As few as three recipients were used to produce twenty SGM founders for four genes. We then demonstrated the powerful multiplex targeting capacity of CRISPR/Cas9. First, two genes on the same chromosome were targeted simultaneously, resulting in three RAG1/RAG2 double-gene mutant (DGM) founders. Next we microinjected forty-five embryos each with five sgRNAs targeting FOXN1, RAG1, RAG2, IL2RG and PRKDC, and transferred them to two recipients. Five founders were produced: one SGM, two DGM, one triple-gene mutant and one quadruple-gene mutant. The present work demonstrates that multiplex embryo transfer and multiplex gene targeting can be used to quickly and efficiently generate mutant rabbit founders. Four lines of SGM (e.g. FOXN1, RAG2, IL2RG, and PRKDC) immunodeficient rabbits, as well as multigenic mutant immunodeficient rabbits have been produced. These animals may prove useful for biomedical research.

Production of immunodeficient rabbits by multiplex embryo transfer and multiplex gene targeting.

Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (-/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by  in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by , as confirmed by histological examination and PCR analysis of  genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.

Jun Song

Center for Advanced Models for Translational Sciences and Therapeutics,
Department of Internal Medicine,
Center for Advanced Models for Translational Sciences and Therapeutics, Department of Internal Medicine

University of Michigan Medical Center

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Jun Song

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Center for Advanced Models for Translational Sciences and Therapeutics,Department of Internal Medicine,Center for Advanced Models for Translational Sciences and Therapeutics, Department of Internal Medicine

University of Michigan Medical Center

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Center for Advanced Models for Translational Sciences and Therapeutics,
Department of Internal Medicine,
Center for Advanced Models for Translational Sciences and Therapeutics, Department of Internal Medicine