Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease

Abstract

Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.

Figure 1. Description of Swine Class I MHC Genes

A) The class I region of swine MHC contains three classical class I genes (SLA-1, −2, −3;), several pseudogenes (SLA-4, −5, and −9) and two class I like genes (SLA-11 and −12). The approximate size of the entire classical class I MHC region is 130kb. The SLA-1 and −3 loci are separated by approximately 65kb and the SLA-2 and −3 loci are separated by about 17kb. B) NCBI accession numbers are shown that are relevant to the alleles of this study. C) The five protein domains of the class I protein are shown with an indication of which gene exon encodes each specific polypeptide region. The β₂m protein is also shown. D) Description of the relative location of the gRNA targets in exon four of the class I gene. E) gRNA target sequences of exon four are shown. Exon four consists of 276 bp in the different alleles, and the class I genes are approximately 3.5 kb long.

Figure 1. Description of Swine Class I MHC Genes

A) The class I region of swine MHC contains three classical class I genes (SLA-1, −2, −3;), several pseudogenes (SLA-4, −5, and −9) and two class I like genes (SLA-11 and −12). The approximate size of the entire classical class I MHC region is 130kb. The SLA-1 and −3 loci are separated by approximately 65kb and the SLA-2 and −3 loci are separated by about 17kb. B) NCBI accession numbers are shown that are relevant to the alleles of this study. C) The five protein domains of the class I protein are shown with an indication of which gene exon encodes each specific polypeptide region. The β₂m protein is also shown. D) Description of the relative location of the gRNA targets in exon four of the class I gene. E) gRNA target sequences of exon four are shown. Exon four consists of 276 bp in the different alleles, and the class I genes are approximately 3.5 kb long.

Figure 1. Description of Swine Class I MHC Genes

A) The class I region of swine MHC contains three classical class I genes (SLA-1, −2, −3;), several pseudogenes (SLA-4, −5, and −9) and two class I like genes (SLA-11 and −12). The approximate size of the entire classical class I MHC region is 130kb. The SLA-1 and −3 loci are separated by approximately 65kb and the SLA-2 and −3 loci are separated by about 17kb. B) NCBI accession numbers are shown that are relevant to the alleles of this study. C) The five protein domains of the class I protein are shown with an indication of which gene exon encodes each specific polypeptide region. The β₂m protein is also shown. D) Description of the relative location of the gRNA targets in exon four of the class I gene. E) gRNA target sequences of exon four are shown. Exon four consists of 276 bp in the different alleles, and the class I genes are approximately 3.5 kb long.

Figure 1. Description of Swine Class I MHC Genes

A) The class I region of swine MHC contains three classical class I genes (SLA-1, −2, −3;), several pseudogenes (SLA-4, −5, and −9) and two class I like genes (SLA-11 and −12). The approximate size of the entire classical class I MHC region is 130kb. The SLA-1 and −3 loci are separated by approximately 65kb and the SLA-2 and −3 loci are separated by about 17kb. B) NCBI accession numbers are shown that are relevant to the alleles of this study. C) The five protein domains of the class I protein are shown with an indication of which gene exon encodes each specific polypeptide region. The β₂m protein is also shown. D) Description of the relative location of the gRNA targets in exon four of the class I gene. E) gRNA target sequences of exon four are shown. Exon four consists of 276 bp in the different alleles, and the class I genes are approximately 3.5 kb long.

Figure 1. Description of Swine Class I MHC Genes

A) The class I region of swine MHC contains three classical class I genes (SLA-1, −2, −3;), several pseudogenes (SLA-4, −5, and −9) and two class I like genes (SLA-11 and −12). The approximate size of the entire classical class I MHC region is 130kb. The SLA-1 and −3 loci are separated by approximately 65kb and the SLA-2 and −3 loci are separated by about 17kb. B) NCBI accession numbers are shown that are relevant to the alleles of this study. C) The five protein domains of the class I protein are shown with an indication of which gene exon encodes each specific polypeptide region. The β₂m protein is also shown. D) Description of the relative location of the gRNA targets in exon four of the class I gene. E) gRNA target sequences of exon four are shown. Exon four consists of 276 bp in the different alleles, and the class I genes are approximately 3.5 kb long.

Figure 2. gRNA-Cas9 Treatment and Flow Sorting of Class I SLA Negative Fibroblast Cells

Following gRNA treatment, two successive rounds of flow cytometry sorting yielded class I negative SLA cells. A representative example of enrichment is shown (A). When used singly or in combination, all three gRNA targeting exon four were capable of producing cells deficient in class I SLA expression (B).

Figure 2. gRNA-Cas9 Treatment and Flow Sorting of Class I SLA Negative Fibroblast Cells

Following gRNA treatment, two successive rounds of flow cytometry sorting yielded class I negative SLA cells. A representative example of enrichment is shown (A). When used singly or in combination, all three gRNA targeting exon four were capable of producing cells deficient in class I SLA expression (B).

Figure 3. Selection of Class I SLA negative Fetal Fibroblast Cells

SCNT of fibroblasts isolated in figure 2 were used to create embryos. 32 days after impregnating a sow with these embryos, three fetuses were collected. Two of the fetuses were well formed and used to create fibroblast cultures. The fibroblasts were stained with a negative isotype control or with and antibody specific for class I SLA. Fetus-3 expressed low levels of SLA protein. Cells derived from Fetus-2 were devoid of class I SLA proteins.

Figure 4. Phenotypic and cDNA Analyses of Class I SLA Deficient Piglets

A) Three piglets, recloned from the SLA negative fetal fibroblast cells isolated in Figure 3, were examined for cell surface expression of class I SLA proteins on the surface of fibroblasts, (all three animals), PBMC (piglets-2 and −3), or cells isolated from the kidney (piglet-1). Corresponding class I SLA positive cells are shown for comparison. Relative binding of class I specific SLA antibodies and an irrelevant isotype control are shown. B) cDNA, prepared from fetus-2 and piglets-1 and −2, were subjected to PCR with primers designed to amplify individual alleles of class I SLA. Sample W represents an identical analysis of the untreated parental, SLA expressing, fibroblasts. The length of the predicted full-length transcript is indicated below each allele name. Samples F, 1, and 2 represent the fetus, and cloned animals 1 and 2 respectively.

Figure 4. Phenotypic and cDNA Analyses of Class I SLA Deficient Piglets

A) Three piglets, recloned from the SLA negative fetal fibroblast cells isolated in Figure 3, were examined for cell surface expression of class I SLA proteins on the surface of fibroblasts, (all three animals), PBMC (piglets-2 and −3), or cells isolated from the kidney (piglet-1). Corresponding class I SLA positive cells are shown for comparison. Relative binding of class I specific SLA antibodies and an irrelevant isotype control are shown. B) cDNA, prepared from fetus-2 and piglets-1 and −2, were subjected to PCR with primers designed to amplify individual alleles of class I SLA. Sample W represents an identical analysis of the untreated parental, SLA expressing, fibroblasts. The length of the predicted full-length transcript is indicated below each allele name. Samples F, 1, and 2 represent the fetus, and cloned animals 1 and 2 respectively.

Figure 5. Lymphocyte Subset Analysis of SLA Expressing and SLA Deficient Pigs

PBMC were isolated from a class I SLA positive animal and two cloned pigs devoid of class I SLA molecules. Cells were incubated with a fluorescent viability dye, and antibodies specific for CD3, CD4, and CD8 molecules. A) Representative histograms demonstrating the gating strategy to select for viable CD3 positive cells. B) CD4 and CD8 expression levels are shown to reveal each T cell subset. An isotype control staining was used to set the gates defining each subset. C) The analysis of panel B was repeated on four separate PBMC isolations from the SLA positive animal and five separate PBMC isolations from the cloned animals (twice for Pig 2 and three times for Pig 3). The means and standard deviations are shown for the various lymphocyte subsets (DN: CD4CD8, DP: CD4⁺CD8⁺, CD4: CD4⁺CD8, CD8: CD4CD8⁺). Unpaired t tests were used to compare the frequencies of each cell type in SLA expressing and SLA deficient animals. P values are shown beneath the graph for comparison of the frequency of each subset between SLA positive and SLA negative animals.