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Expanded Materials and Methods, Supplemental Figures, and Supplemental Tables Figure S1. Flow cytometric analysis of peripheral blood from SS, SS.BN3, SSIL2Rγ, and SS.BN3IL2Rγ rats. Figure S2.SH2B3 specifically marks CD31+ blood vessels, but not LYVE1+ lymphatic vessels. Table S1. Peripheral blood analysis of SS, SS.BN3, SSIL2Rγ and SS.BN3IL2Rγ rats. Table S2. Genes located in the overlapping portions of the rat chromosome 3 cancer QTLs. Table S3. Variants that cosegregate between tumor resistant (BN and Cop) and tumor susceptible (ACI, F344, and SS) rat strains.

The majority of causative variants in familial breast cancer remain unknown. Of the known risk variants, most are tumor cell autonomous, and little attention has been paid yet to germline variants that may affect the tumor microenvironment. In this study, we developed a system called the Consomic Xenograft Model (CXM) to map germline variants that affect only the tumor microenvironment. In CXM, human breast cancer cells are orthotopically implanted into immunodeficient consomic strains and tumor metrics are quantified (e.g., growth, vasculogenesis, and metastasis). Because the strain backgrounds vary, whereas the malignant tumor cells do not, any observed changes in tumor progression are due to genetic differences in the nonmalignant microenvironment. Using CXM, we defined genetic variants on rat chromosome 3 that reduced relative tumor growth and hematogenous metastasis in the SS.BN3IL2Rγ consomic model compared with the SSIL2Rγ parental strain. Paradoxically, these effects occurred despite an increase in the density of tumor-associated blood vessels. In contrast, lymphatic vasculature and lymphogenous metastasis were unaffected by the SS.BN3IL2Rγ background. Through comparative mapping and whole-genome sequence analysis, we narrowed candidate variants on rat chromosome 3 to six genes with a priority for future analysis. Collectively, our results establish the utility of CXM to localize genetic variants affecting the tumor microenvironment that underlie differences in breast cancer risk. Cancer Res; 74(22); 6419–29. ©2014 AACR.

Sweet potato (Ipomoea batatas (L.) Lam.) is one of the most important root crops in the world. Initial formation and development of storage roots (SRs) are key factors affecting its yields. In order to study the molecular mechanism and regulatory networks of the SRs development process, we have analyzed root transcriptomes between the high and low starch content sweet potato accessions at three different developmental stages. In this study, we assembled 46,840 unigenes using Illumina paired-end sequencing reads and identified differentially expressed genes (DEGs) between two accessions. The numbers of DEGs were increased with the development of SRs, indicating that the difference between two accessions is enlarging with the maturation. DEGs were mainly enriched in starch biosynthesis, plant hormones regulatory, and genetic information processing pathways. Then, expression patterns of DEGs that are most significant and starch biosynthesis related were validated using qRT-PCR. Our results provide valuable resources to future study on molecular mechanisms of SRs development and candidate genes for starch content improvement in sweet potato.

Figure S1. Diploid Ipomoea trifida and sweetpotato. (a) Whole plant of Y22, showing storage root (SR) formation. Y22 is a clone of diploid I. trifida seeds (CIP No: PC98_1 (698014), female parent 2X P96124.5, male parent PC). (b) Whole plant of Y25, which does not form SRs. Y25 is a clone of diploid I. trifida seeds (CIP No: 696153, female parent 2X 6.1, male parent OP). (c) Transverse and longitudinal sections of a pencil root (PR) from Y25 and a SR from Y22; the cortex can be easily stripped from the Y22 SR. (d) F1 progeny (clone 3–11) of Y22, which also has strong SR development. (e) The SR of sweetpotato var. Xushu22. Scale bar: 2 cm. Figure S2. K-mer analysis for estimating the genome size of I. trifida. K = 17. The X-axis shows the depth, and the Y-axis represents the frequency at each depth. Figure S3. Heterozygosity assessment using a fitting curve. The light blue curve is consistent with the heterozygosity of the genome. Therefore, the heterozygosity is 2.20%. Figure S4. SSR identification of true F1 individuals. M represents the marker. Y25 was the female parent, and Y22 was the male parent. 3–11, 1–6, 2–6, 4–1, 2–3 and 2–7 were the F1 individuals. In the electrophoretic bands, any of the bands with Y22 existed on the basis of the Y25 bands in the progeny is true hybrid. Figure S5. The high-density genetic map of I. trifida. Figure S6. The fifteen pseudochromosomes of I. trifida. The scaffolds were anchored to pseudochromosomes according to the locations of markers from the constructed linkage map. The blue pillars represent the chromosomes, which each consist of multiple scaffolds. The green pillars represent the fifteen linkage groups, and the grey lines link the markers from the linkage groups to the physical locations on the chromosomes. Figure S7. GC content and mean sequence depth calculated with a 10 k non-overlapping sliding window. The x-axis represents the GC content, and the y-axis represents the average depth with 10-kb non-overlapping sliding windows. The histogram at right represents the average depth distribution, while the histogram above represents the GC content distribution of the I. trifida genome. Figure S8. GC content and mean sequence depth of the I. trifida genome calculated with a 10 k non-overlapping sliding window. The x-axis represents the GC content, and the y-axis represents the density of GC content. Figure S9. Gene set evidence supports statistics. The blue circle represents the 29,728 genes predicted de novo, the light blue circle represents the 24,109 genes predicted by RNA evidence, and the red circle represents the 25,618 genes predicted by homology from seven species including Arabidopsis thaliana, Beta vulgaris, Capsicum annuum, Sesamum indicum, Solanum lycopersicum, Solanum tuberosum and Vitis vinifera. Figure S10. Insertion time distribution of LTR-RTs of I. trifida and I. nil. The y-axis represents the copy numbers of LTR-RTs and the x-axis represents the insertion time of LTR-RTs. We performed alignment of the sequences between the 5′ and 3′ LTRs using MUSCLE (v3.8.31, http://www.drive5.com/muscle ). LTR insertion time (T) was calculated with the formula T = k/2r (divergence between LTRs / substitution per site per year, r = 1.05E-8). Figure S11. Collinear blocks between I. trifida and I. nil. Different colours represent different chromosomes. Figure S12. Collinear blocks between I. trifida and the haplotype-resolved I. batatas genome. Different colours represent different chromosomes. Itr: I. trifida, Ib: I. batatas. Figure S13. Venn diagrams of differentially expressed gene numbers when comparing groups S1 vs S0, S2 vs S0 and S3 vs S0. (a) Venn diagram of all differentially expressed genes, including up- and downregulated genes. (b) Venn diagram of differentially downregulated genes. (c) Venn diagram of differentially upregulated genes in the three comparison groups. The differentially upregulated genes were defined using DESeq with Padj

Table S1. Survey statistic results. Table S2. Sequencing data statistics. Table S3. Statistics of assembly results with only Illumina sequencing data. Table S4. Statistics of assembly results after extension with PacBio RS II data. Table S5. SSR primer used for identification of true F1 hybrids. Table S6. Statistical information of genetic linkage groups. Table S7. Coverage statistics of the I. trifida genome. Table S8. EST sequence evaluation results. Table S9. CEGMA evaluation results. Table S10. RNA-seq data used for annotation. Table S11. Statistical results of gene functional annotations. Table S12. Statistical results of non-coding RNAs. Table S13. Statistical results of repeated classification. Table S14. Summary of the plant species and assemblies/gene models used in this study. Table S15. Comparison of repeat contents between I. trifida and I. nil. Table S16. Statistics of LTR numbers. Table S17. Lengths of syntenic blocks and block repeat sequences. Table S18. Chromosome duplication test results for Y22. Table S19. KEGG enrichment results for the genes expanded in I. trifida. Table S20. KEGG enrichment results for the genes contracted in I. trifida. Table S21. Sequencing data statistics of RNA from S0 to S3. Table S22. KEGG pathways of upregulated genes. Table S23. Blast results of the specific protein sporamin in the I. trifida assembly. Table S24. KEGG pathways of downregulated genes. Table S25. QTL mapping results. Table S26. Results of BMY11 microarray hybridization during the process of SR development in sweetpotato var. Guangshu87. Table S27. Blast results of BMY11 in the full-length transcripts of sweetpotato var. Xushu18. Table S28. qPCR primers used to amplify BMY11. (DOCX 75 kb)

For modeling solidification process of casting accurately, a reliable heat transfer boundary condition data is required. In this paper, an inverse conduction model was established to determine the heat flux and heat transfer coefficient at the metal–sand mold interface for cylindrical casting in the lost foam process. The numerically calculated temperature was compared with analytic solution and simulation solution obtained by commercial software ProCAST to investigate the accuracy of heat conduction model. The instantaneous cast and sand mold temperatures were measured experimentally and these values were used to determine the interfacial heat transfer coefficient (IHTC). The IHTC values during lost foam casting were shown to vary from 20 to 800 W m−2 K−1. Additionally, the characteristics of the time-varying IHTC have also been discussed in this study.

The majority of causative variants in familial breast cancer remain unknown. Of the known risk variants, most are tumor cell autonomous, and little attention has been paid yet to germline variants that may affect the tumor microenvironment. In this study, we developed a system called the Consomic Xenograft Model (CXM) to map germline variants that affect only the tumor microenvironment. In CXM, human breast cancer cells are orthotopically implanted into immunodeficient consomic strains and tumor metrics are quantified (e.g., growth, vasculogenesis, and metastasis). Because the strain backgrounds vary, whereas the malignant tumor cells do not, any observed changes in tumor progression are due to genetic differences in the nonmalignant microenvironment. Using CXM, we defined genetic variants on rat chromosome 3 that reduced relative tumor growth and hematogenous metastasis in the SS.BN3IL2Rγ consomic model compared with the SSIL2Rγ parental strain. Paradoxically, these effects occurred despite an increase in the density of tumor-associated blood vessels. In contrast, lymphatic vasculature and lymphogenous metastasis were unaffected by the SS.BN3IL2Rγ background. Through comparative mapping and whole-genome sequence analysis, we narrowed candidate variants on rat chromosome 3 to six genes with a priority for future analysis. Collectively, our results establish the utility of CXM to localize genetic variants affecting the tumor microenvironment that underlie differences in breast cancer risk. Cancer Res; 74(22); 6419–29. ©2014 AACR.

One of the most powerful strategies to investigate biology we have as scientists, is the ability to transfer genetic material in a controlled and deliberate manner between organisms. When applied to livestock, applications worthy of commercial venture can be devised. Although initial methods used to generate transgenic livestock resulted in random transgene insertion, the development of SCNT technology enabled homologous recombination gene targeting strategies to be used in livestock. Much has been accomplished using this approach. However, now we have the ability to change a specific base in the genome without leaving any other DNA mark, with no need for a transgene. With the advent of the genome editors this is now possible and like other significant technological leaps, the result is an even greater diversity of possible applications. Indeed, in merely 5 years, these ‘molecular scissors’ have enabled the production of more than 300 differently edited pigs, cattle, sheep and goats. The advent of genome editors has brought genetic engineering of livestock to a position where industry, the public and politicians are all eager to see real use of genetically engineered livestock to address societal needs. Since the first transgenic livestock reported just over three decades ago the field of livestock biotechnology has come a long way—but the most exciting period is just starting.

We describe a fundamentally novel feat of animal genetic engineering: the precise and efficient substitution of an agronomic haplotype into a domesticated species. Zinc finger nuclease in-embryo editing of the RELA locus generated live born domestic pigs with the warthog RELA orthologue, associated with resilience to African Swine Fever. The ability to efficiently achieve interspecies allele introgression in one generation opens unprecedented opportunities for agriculture and basic research.

The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.

Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate recombination of DNA into the pig genome. This often results in animals containing concatemeric arrays of transgenes that complicate characterization and can impair long-term transgene stability and expression. This is inconsistent with regulatory guidance for transgenic livestock, which also discourages the use of selection markers, particularly antibiotic resistance genes. We demonstrate that the Sleeping Beauty (SB) transposon system effectively delivers monomeric, multi-copy transgenes to the pig embryo genome by pronuclear injection without markers, as well as to donor cells for founder generation by cloning. Here we show that our method of transposon-mediated transgenesis yielded 38 cloned founder pigs that altogether harbored 100 integrants for five distinct transposons encoding either human APOBEC3G or YFP-Cre. Two strategies were employed to facilitate elimination of antibiotic genes from transgenic pigs, one based on Cre-recombinase and the other by segregation of independently transposed transgenes upon breeding.

Reprogramming of cellular identity using exogenous expression of transcription factors (TFs) is a powerful and exciting tool for tissue engineering, disease modeling, and regenerative medicine. However, generation of desired cell types using this approach is often plagued by inefficiency, slow conversion, and an inability to produce mature functional cells. Here, we show that expression of constitutively active SMAD2/3 significantly improves the efficiency of induced pluripotent stem cell (iPSC) generation by the Yamanaka factors. Mechanistically, SMAD3 interacts with reprogramming factors and co-activators and co-occupies OCT4 target loci during reprogramming. Unexpectedly, active SMAD2/3 also markedly enhances three other TF-mediated direct reprogramming conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons, highlighting broad and general roles for SMAD2/3 as cell-reprogramming potentiators. Our results suggest that co-expression of active SMAD2/3 could enhance multiple types of TF-based cell identity conversion and therefore be a powerful tool for cellular engineering.

We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10-50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.

Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) genome editing technology enables site directed engineering of the genome. Here we demonstrate for the first time that both TALEN and ZFN injected directly into pig zygotes can produce live genome edited pigs. Monoallelic as well as heterozygous and homozygous biallelic events were identified, significantly broadening the use of genome editor technology in livestock by enabling gene knockout in zygotes from any chosen mating.

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.

Since catalytic technology is the key technology for petroleum refining and petrochemical processing and catalyst is the core of catalytic technology, the development of new catalysts would have a great impact on petroleum refining and petrochemical industry. After entering the 21st century, there will be a lot of challenges on China's agenda in the areas of energy security and chemicals production. An effective way to solve these problems is to uninterruptedly develop new catalytic materials and technologies geared to the deep processing of crudes, the production of clean fuels, the production of light olefins and the effective production of green chemicals.

Template-driven HTML documents possess an implicit, fixed schema denoting concepts and their relationships in a hierarchical fashion. Discovering this schema remains a relatively unexplored problem. By exploiting a key observation that semantically related items in HTML documents exhibit spatial locality, we develop an algorithm for automatically partitioning them into tree-like semantic structures which expose the implicit schema.

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100 000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10–9. Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene’s encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%–50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

Genetic enhancement of livestock productivity and welfare are major goals of breeding and genetics programs. However, the introgression of desirable alleles across breeds is slow and inaccurate. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement of a diversity of livestock breeds. Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. We found that TALEN could be easily manufactured and that 64% displayed activity in swine and cattle primary fibroblasts, with cleavage of 1.5 to 45% of chromosomes in cell populations, as measured by Surveyor assay. Clonal isolation and sequencing revealed that up to 84% of cells contained at least one modified allele, with up to 24% of cells containing biallelic or homozygous chromosomal modification. Co-transfection of a customized TALEN pair with a template containing a specific allele was effective at the nonmeiotic introgression of quantitative trait into naïve cattle breeds. We will also describe the repair of 2 recently described embryonic lethal mutations that are segregating in important dairy cattle breeds (JH1 and HH1). Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in 27 to 75% of embryos analysed (n = 4–59), nearly half of which (8/19) harbored biallelic modification. We will present data describing efforts towards gene conversion by direct injection of livestock embryos. Finally, we will present alternative strategies for the incorporation of gene editing in livestock production systems by cloning or embryo treatment.

TALEN-induced double-strand breaks can be used for gene inactivation via repair by non-homologous end joining (NHEJ) or to stimulate homologous recombination (HR). HR can be used to introduce custom genetic modifications or to introgress naturally occurring alleles. We found that over 65% of custom-designed TALENs displayed activity in pig and cattle fibroblasts, with a typical percentage of indel positive chromosomes ranging from 20 to 45%. Isolation of individual clones with mono- and biallelic modifications to targeted loci was extremely efficient (up to 84 and 24% of clones, respectively) and could be accomplished without the aid of selection. Co-transfection of TALENs with a homologous repair template enabled precise insertion of a novel restriction site in nearly 40% of treated cells, with surprising levels of homozygosity. To prove that gene-edited Ossabaw swine cells were suitable for the generation of animals by cloning, we pooled colonies harboring both monoallelic and biallelic TALEN-induced frame-shift mutations in the swine low-density lipoprotein receptor (LDLR) and used them as nuclear donors for chromatin transfer. Pregnancy was established in 7/9 transfers, and 6 pregnancies were carried to term, resulting in the live birth of 18 piglets. Pigs heterozygous and homozygous for TALEN-induced mutations are being investigated as models of familial hypercholesterolemia (FH). We have additionally targeted the same locus for HR using a specified inactivating mutation. Fibroblasts heterozygous and homozygous for a specific 4-bp insertion into LDLR were created by allele introgression and have been cloned by chromatin transfer, demonstrating that gene editing can be used to create precise, swine knock-ins in a single generation. Allele introgression is also critical to livestock genetics, where crossbreeding has been a staple of breeding programs. Although major effect alleles for enhancing productivity and animal welfare have been discovered, the introgression of low-frequency alleles by traditional breeding is slow and inaccurate, involving recombination across the entire genome. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement in a diversity of livestock breeds. Co-transfection of a TALEN pair with a template containing a specific, naturally occurring allele was effective at the non-meiotic introgression of quantitative traits into the genome of cells from naïve cattle breeds, now being used to create founders by cloning. We will also present progress towards gene conversion by direct injection of livestock embryos. Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in up to 75% of embryos analysed, nearly half of which harbored biallelic modification. We will present alternative strategies for the incorporation of gene editing into livestock genetic improvement programs by either cloning or embryo treatment.

Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA-binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. TALEN-induced double-strand breaks can be used for gene inactivation via repair by nonhomologous end joining (NHEJ) or to stimulate homologous recombination with a user-specified template to create custom-designed disease alleles. Transfection of multiple TALEN pairs targeting the same chromosome can also produce chromosomal rearrangements such as large inversions and deletions. As genetic modification of primary pig fibroblast followed by cloning is the primary method used to generate transgenic swine, we tested our capacity to mimic multiple common mutation types in primary fibroblasts using TALEN. Either plasmids or mRNA encoding TALEN was transfected into 500 000 to 750 000 fibroblasts by nucleofection followed by 3 days of culture at 30°C before analysis of activity. With this approach, we found that over 65% of custom-designed TALEN displayed activity in pig fibroblasts with typical percentage of indel positive chromosomes ranging from 20 to 45%. Individual clones were derived by either co-selection with an unlinked selection transposon or by dilution cloning. Colonies with mono- and biallelic modification to the target loci were readily identified (up to 84 and 24%, respectively) and could be accomplished without the aid of selection. Co-transfection of 2 TALEN pairs directed to the same chromosome resulted in deletion or inversion of the intervening sequence in 10 and 4% of isolated colonies, respectively. In addition, co-transfection of TALEN with a homologous repair template enabled precise insertion of a novel restriction site in nearly 40% of treated cells, 3% of which were homozygous (n = 184 colonies). To prove that modified cells were suitable for generation of animals, we pooled several colonies of Ossabaw fibroblasts harboring TALEN-induced frame-shift mutations in the swine low-density lipoprotein receptor (LDLR) and used them as nuclear donors for cloning. Pregnancy was established in 7/9 transfers (100–150 embryos per transfer), and 6 pregnancies were carried to term, resulting in the live birth of 18 piglets. Three monoallelic frame-shift genotypes were observed among 9 piglets; as expected, each genotype was represented in the parental cell pool. The remaining 9 animals displayed an identical biallelic mutant genotype and are being investigated as a knockout model of familial hypercholesterolemia (FH). Finally, fibroblasts homozygous for a specified inactivating mutation were created by allele introgression and were cloned, demonstrating that gene editing can be used to create precise swine knock-ins in a single generation.