Inclusion of homologous DNA in nuclease-mediated gene targeting facilitates a higher incidence of bi-allelically modified cells.
ABSTRACT: Recent advancements in gene editing techniques have increased in number and utility. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the high efficiency of gene editing. We have previously produced cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFNs) in combination with donor DNA containing a total homology length of 1600 bp (800-bp homology on each arm). Our next aim was to introduce the targeted disruption of alpha-1,3-galactosyltransferase (GGTA1) in the CMAH KO genetic background and evaluate the effect of donor DNA homology length on meganuclease-mediated gene targeting.Zinc-finger nucleases from a previous CMAH KO experiment were used as a proof of concept to identify a correlation between the length of donor DNA homology and targeting efficiency. Based on those results, experiments were designed to use transcription activator-like effector nucleases (TALENs) to generate bi-allelically modified GGTA1 cells using donor DNAs carrying various lengths of homology. Donor DNA was designed to symmetrically flank the predicted cleavage sites in CMAH and GGTA1 for both ZFN and TALEN cleavage sites, respectively. For both genes, the length of total homology ranged from 60 to 1799 bp. Sialyltransferase gene expression profiles were evaluated in CMAH and GGTA1 double KO pig cells and were compared to wild-type and CMAH KO cells.Introduction of donor DNA with ZFNs demonstrated that small amounts of homology (60 bp) could facilitate homology-directed repair during ZFN-mediated targeting of CMAH; however, donor DNA with longer amounts of homology resulted in a higher frequency of homology-directed repair. For the GGTA1 KO experiments that used TALENs and donor DNA, donor DNA alone did not result in detectable bi-allelic conversion of GGTA1. As the length of donor DNA increased, the bi-allelic disruption of GGTA1 increased from 0.5% (TALENs alone, no donor DNA present) to a maximum of 3% (TALENs and donor DNA with total homology of 1799 bp). Inclusion of homologous donor DNA in TALEN-mediated gene targeting facilitated a higher incidence of bi-allelically modified cells. Using the generated cells, we were able to demonstrate the lack of GGTA1 expression and the decrease in gene expression sialyltransferase-related genes.The approach of using donor DNA in conjunction with a meganuclease can be used to increase the efficiency of gene targeting. The gene editing methods can be applied to other genes as well as other mammalian systems. Additionally, gene expression analysis further confirms that the CMAH/GGTA1 double KO pigs can be a valuable source for the study of pig-to-human xenotransplantation.
Project description:Pigs are ideal organ donors for xenotransplantation and an excellent model for studying human diseases, such as neurodegenerative disease. Transcription activator-like effector nucleases (TALENs) are used widely for gene targeting in various model animals. Here, we developed a strategy using TALENs to target the GGTA1, Parkin and DJ-1 genes in the porcine genome using Large White porcine fibroblast cells without any foreign gene integration. In total, 5% (2/40), 2.5% (2/80), and 22% (11/50) of the obtained colonies of fibroblast cells were mutated for GGTA1, Parkin, and DJ-1, respectively. Among these mutant colonies, over 1/3 were bi-allelic knockouts (KO), and no off-target cleavage was detected. We also successfully used single-strand oligodeoxynucleotides to introduce a short sequence into the DJ-1 locus. Mixed DJ-1 mutant colonies were used as donor cells for somatic cell nuclear transfer (SCNT), and three female piglets were obtained (two were bi-allelically mutated, and one was mono-allelically mutated). Western blot analysis showed that the expression of the DJ-1 protein was disrupted in KO piglets. These results imply that a combination of TALENs technology with SCNT can efficiently generate bi-allelic KO pigs without the integration of exogenous DNA. These DJ-1 KO pigs will provide valuable information for studying Parkinson's disease.
Project description:Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target ?1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.
Project description:Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated ?-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.
Project description:BACKGROUND:Antipig antibodies are a barrier to clinical xenotransplantation. We evaluated antibody binding of waitlisted renal transplant patients to 3 glycan knockout (KO) pig cells and class I swine leukocyte antigens (SLA). METHODS:Peripheral blood mononuclear cells from SLA identical wild type (WT), ?1, 3-galactosyltransferase (GGTA1) KO, GGTA1/ cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) KO, and GGTA1/ CMAH /b1,4 N-acetylgalactosaminyl transferase (B4GalNT2) KO pigs were screened for human antibody binding using flow cytometric crossmatch (FCXM). Sera from 820 patients were screened on GGTA1/CMAH/B4GalNT2 KO cells and a subset with elevated binding was evaluated further. FCXM was performed on SLA intact cells and GGTA1/SLA class I KO cells after depletion with WT pig RBCs to remove cell surface reactive antibodies, but leave SLA antibodies. Lastly, human and pig reactive antibodies were eluted and tested for cross-species binding and reactivity to single-antigen HLA beads. RESULTS:Sequential glycan KO modifications significantly reduce antibody binding of waitlisted patients. Sera exhibiting elevated binding without reduction after depletion with WT RBCs demonstrate reduced binding to SLA class I KO cells. Human IgG, eluted from human and pig peripheral blood mononuclear cells, interacted across species and bound single-antigen HLA beads in common epitope-restricted patterns. CONCLUSIONS:Many waitlisted patients have minimal xenoreactive antibody binding to the triple KO pig, but some HLA antibodies in sensitized patients cross-react with class I SLA. SLA class I is a target for genome editing in xenotransplantation.
Project description:We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [?2,3-sialyltransferase (?2,3ST) and ?2,6-sialyltransferase (?2,6ST)] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls. The elevated level of sialic-acid-containing glycoconjugate epitopes was due to the low level of ?-Gal in heterozygote GalT KO livers. Furthermore, proteomics analysis showed that heterozygote GalT KO pigs had a higher expression of NAD+-isocitrate dehydrogenase (IDH), which is related to the CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme reaction. These findings suggest the deficiency of GGTA1 gene in pigs results in increased production of N-glycolylneuraminic acid (Neu5Gc) due to an increase of ?2,6-sialyltransferase and a CMAH cofactor, NAD+-IDH. This indicates that Neu5Gc may be a critical xenoantigen. The deletion of the CMAH gene in the GalT KO background is expected to further prolong xenograft survival.
Project description:Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The ?4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes.The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/?4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons.Cells from GGTA1/CMAH/?4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Non-human primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased non-human primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/?4GalNT2.Inactivation of the ?4GalNT2 gene reduces human and non-human primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of non-human primates toward GGTA1-/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/?4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the non-human primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/?4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.
Project description:Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as <i>GGTA1</i>, <i>CMAH</i>, and <i>B4GALNT2</i>, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting <i>GGTA1</i>, <i>CMAH,</i> and <i>B4GALNT2</i> into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting <i>GGTA1</i> and <i>CMAH</i> with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when <i>GGTA1</i>, <i>CMAH</i>, and <i>B4GALNT2</i> were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of <i>GGTA1</i>/<i>CMAH</i> double-edited pigs and <i>GGTA1</i>/<i>CMAH/B4GALNT2</i> triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.
Project description:When hyperacute rejection is avoided by deletion of Gal expression in the pig, delayed xenograft rejection (DXR) becomes a major immunologic barrier to successful xenotransplantation. This study was to investigate the potential antigens involved in DXR. We isolated primary renal microvascular endothelial cells (RMEC) and aortic endothelial cells (AEC) from a GGTA1/CMAH double-knockout (DKO) pig (and a GGTA1-KO pig) and immunized cynomolgus monkeys with both of these cells. After sensitization, monkey serum antibody binding and cytotoxicity to RMEC was significantly higher than to AEC(p?<?0.05), suggesting that RMEC are more immunogenic than AEC. Transcriptome sequencing of GGTA1/CMAH DKO pigs indicated that the expression of 1,500 genes was higher in RMEC than in AEC, while expression of 896 genes was lower. Next, we selected 101 candidate genes expressed only in pig RMEC, but not in pig AEC or in monkey or human RMEC. When these genes were knocked out individually in GGTA1/CMAH DKO RMEC, 32 genes were associated with reduced antibody binding, indicating that these genes might be primary immunologic targets involved in DXR. These genes may be important candidates for deletion in producing pigs against which there is a reduced primate immune response in pig kidney xenograft.
Project description:A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediated injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein, we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury.Wild type (WT), ASGR1, GGTA1, and GGTA1CMAH knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1, GGTA1, and GGTA1 CMAH pigs.GGTA1, CMAH LSECs exhibited reduced levels of human platelet binding in vitro when compared with GGTA1 and WT LSECs. In a continuous perfusion model, GGTA1 CMAH livers consumed fewer human platelets than GGTA1 and WT livers. GGTA1 CMAH livers also consumed fewer human platelets than ASGR1 livers in a single-pass model.Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation.
Project description:Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-?1,3-galactose (?Gal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including ?(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of ?Gal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both ?Gal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet.