Wenfang Tan

In vivo genome editing using a high-efficiency TALEN system

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.

Efficient TALEN-mediated gene knockout in livestock

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.

Efficient nonmeiotic allele introgression in livestock using custom endonucleases

Significance Selective breeding has long been practiced to enrich for desirable DNA variation that influences livestock traits. We demonstrate that genetic variants can be directly introgressed into livestock genomes using a modified transcription activator-like effector nuclease system. The transient exposure of livestock cells to sequence-targeted editors stimulates homology-directed repair to levels that eliminate the need for transgene-dependent selection. Use of oligonucleotide template enables efficient single nucleotide changes to the genome and permits the transmission of both natural and novel DNA sequence variants into naïve livestock breeds and species. Gene editing offers a powerful method for accelerating the genetic improvement of livestock for food and also for developing swine as a resource for regenerative medicine and models of human disease. 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.

Live pigs produced from genome edited zygotes

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.

Strategies for selection marker-free swine transgenesis using the Sleeping Beauty transposon system

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.

Mammalian interspecies substitution of immune modulatory alleles by genome editing

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.

Editing livestock genomes with site-specific nucleases

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 100000-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 genes 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.

CXM: A New Tool for Mapping Breast Cancer Risk in the Tumor Microenvironment

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.BN3(IL2Rγ) consomic model compared with the SS(IL2Rγ) 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.BN3(IL2Rγ) 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.

Constitutively Active SMAD2/3 Are Broad-Scope Potentiators of Transcription-Factor-Mediated Cellular Reprogramming

Summary 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.