Site-specifically Editing Of Porcine Genome By TALEN-mediated Gene Targeting

Gene targeting technology emerged in the1980s, and has greatly advancedbiomedical research and development biology. Over the past decades, geneticresearch has developed from initial sequencing and mapping to analyzing genefunction and pathogenic mechanism. This development has resulted in tremendousbreakthroughs in modern biology and medical research. Gene targeting technologyhas been conducted in mice for a long time because their embryonic stem cells canproliferate infinitely in vitro and they can form chimeric progeny. However, mousemodels cannot accurately reflect real pathological processes and diseasedevelopment when used to mimic human diseases. These models even show oppositeresults. Thus, researchers have been investigating large animals with similaranatomy and physiology to humans for use as disease models in future studies.Large animal models, particularly pig models, have similar physiological,biochemical, metabolic, and nutrient characteristics to human beings. Therefore, pigis regarded as one of the most ideal genetically modified animal models. Since thesuccess of cloning pigs using somatic cell nuclear transfer by PPL Company in2000,many breakthroughs have been achieved by genetically modified pigs inxenotransplantation research, human disease models, and agricultural breedimprovement. However, researchers mainly generate genetically modified pigsthrough gene targeting and nuclear transfer of somatic cells because of the lack ofporcine embryonic stem cells. Somatic cells have limited reproducibility in vitro,and conventional gene targeting using homologous recombination is inefficient.Thus, few gene targeting pig models have been reported. Improving gene targetingtechnology is the key to develop research on genetically modified pigs. Gene targeting technology, such as zinc-finger nucleases (ZFNs), transcriptionactivator-like effector nucleases (TALENs), and clustered-regularly interspaced shortpalindromic repeat (CRISPR)/CRISPR-associated (CRISPR/Cas9) system, has obtainedsignificant breakthroughs in gene targeting. Thus, efficient tools have been developed forgene targeting research in pigs. However, ZFN is inefficient and expensive, which hinder itsapplication in pig gene targeting research. CRISPR/Cas9produces severe off-targetingeffects, which limit its applications in somatic cell targeting and nuclear transfer. Therefore,TALEN is an ideal gene targeting technology because of its high targeting efficiency, lowprice, and absence of off-target effects.TALEN is an artificial nuclease that is fused by the binding domain of theTALE protein and nuclease domain of the Fokl protein. TALENs targeting any DNAsequence can be constructed using unit assembly. TALENs recognize and cut thespecific gene locus, and induce the double-strand break of the targeting DNAsequence. Thus, we can achieve gene knockout, knock-in, or specific point mutationusing homologous recombination repair or end connection of non-homologousrecombination. Gene targeting modification can be efficiently improved. TALEN hasbeen widely used in eukaryotic cells, mice, rats, zebrafish, pigs, cattles, and otheranimal models. TALEN is a simple method with high targeting efficiency and nooff-target effects. This study aimed to use TALEN in genetically modified pigs.In this study, we first constructed TALEN vectors based on the preference ofmammalian codon, and built TALEN systems based on the reported Golden GateTALEN construction suitable for mammals. We developed a single-strand annealing(SSA) system using EGFP fluorescent protein and DNA single-strand renaturationtechnology. This system can validate easily and rapidly the activity of theconstructed TALENs. Simultaneously, we generated an in vivo activity detectionsystem using a T7endonuclease, which can recognize and cleave a DNAheterodimer. To validate the TALEN system established in this study, six TALENtargets targeting the pROSA26locus were designed and tested using an SSA assayand T7endonuclease I assay. Results show that the targeting efficiency mediated by TALENs in mammalian cells significantly improved. We applied this targetingmethod, including gene knockout, gene knock-in, and point mutation, to modifyefficiently and accurately the genome of Chinese mini pig.In gene knockout, we designed TALENs targeting the seventh exon of theporcine DMD gene and third exon of the porcine WRN gene. After transfection intoporcine fetal fibroblasts with G418selection, we successfully obtained DMDknockout cells and WRN knockout cells. These cells lay the foundation to establishthe Duchenne and progeria gene-modified porcine models in the future. In geneknock-in, we designed TALENs targeting the porcine ISL1gene, and alsoconstructed the gene-targeting vector pFlexibleDT-ISL1-CreTd. Afterco-transfection into porcine fetal fibroblasts with puro selection, we obtainedISL1-targeted cells with high efficiency of up to24%. In point mutation, given thatporcine insulin has only one amino acid different from human insulin, we designedTALENs targeting the A30position of the β-chain in the porcine insulin gene. Wealso synthesized a single-stranded DNA with89bp. After transfection into porcinefetal fibroblasts with G418selection, we obtained targeted cells, in which the alaninewas successfully replaced with threonine, and humanized porcine insulin modelswere generated using the somatic cell nuclear transfer approach.This study is the first to perform TALEN-targeting technology for the precisemodification of the porcine endogenous gene. We successfully and efficientlyachieved gene knockout, knock-in, and point mutation. The achievement of thisapproach could provide a platform for generating gene-modified porcine modelswith high efficiency. This approach could also contribute in generatinggene-modified porcine models with high economic value, improved agriculturalbreeding, and important medical applications.