Abstract
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.
Highlights
Immunodeficient mice carrying mutations in genes that are involved in lymphocyte development and/ or functions, such as FOXN1, IL2RG, RAG1 or RAG2, and PRKDC, are widely used in biomedical research[1]
Production of gene targeted transgenic (GTT) rabbits has been a challenge, until recent advent of gene editing nucleases, including ZFN, TALEN, and CRISPR/Cas[9]. These customizable nucleases are efficient in generating double strand breaks (DSBs), which may lead to a functional knockout (KO) of the targeted gene when the DSBs are repaired by non-homologous end joining (NHEJ), or be used to integrate a DNA sequence at a specific locus through homology directly repair (HDR)[5]
The fact that single guide RNA (sgRNA) can be designed and synthesized makes it possible to use multiple sgRNAs at the same time to achieve multigenic targeting, which has been demonstrated in cells and in transgenic animal production[8,13,14,15]
Summary
Immunodeficient mice carrying mutations in genes that are involved in lymphocyte development and/ or functions, such as FOXN1, IL2RG, RAG1 or RAG2, and PRKDC, are widely used in biomedical research[1]. Production of gene targeted transgenic (GTT) rabbits has been a challenge, until recent advent of gene editing nucleases, including ZFN, TALEN, and CRISPR/Cas[9] (interchangeably referred to as “Cas9”) These customizable nucleases are efficient in generating double strand breaks (DSBs), which may lead to a functional knockout (KO) of the targeted gene when the DSBs are repaired by non-homologous end joining (NHEJ), or be used to integrate a DNA sequence at a specific locus (e.g. knock-in) through homology directly repair (HDR)[5]. The fact that sgRNAs can be designed and synthesized makes it possible to use multiple sgRNAs at the same time to achieve multigenic targeting, which has been demonstrated in cells and in transgenic animal production[8,13,14,15] This multiplex targeting capacity is useful when the targeted genes are closely positioned on the same chromosome (e.g. RAG1 and RAG2). We selected several major immunodeficient-causing genes, FOXN1, RAG1, RAG2, IL2RG, and PRKDC, to demonstrate the applicability of multiplex embryo transfer and multiplex gene targeting for production of GTT rabbits, and to develop rabbit models of immunodeficiency
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