새롭게 부상하는 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) 9 유전자 편집 기술은 장기 이식(organ transplantation)과 같은 생의학 연구(biomedical research)와 동물 산업에 대한 전통적인 접근 방식을 빠르게 변화시키고 있다. 돼지 생식 및 호흡기 증후군 바이러스(porcine reproductive and respiratory syndrome virus; PRRSV)와 전염성 위장염 바이러스 (transmissible gastroenteritis coronavirus; TGEV)는 돼지 산업에 막대한 경제적 손실을 초래하는 치명적인 바이러스이다. 바이러스의 숙주 수용체 단백질 CD163과 pAPN에 대한 이중 유전자 녹아웃(double knock-out; DKO) 돼지는 PRRSV와 TEGV에 내성을 나타내었으며, 정상(wild-type; WT) 돼지와 비교할 때 성장과 생식 특성의 차이가 없었다. 이러한 결과는 경제 동물 돼지에 CRISPR-Cas9 매개 유전자 편집 기술을 적용하여 바이러스 저항성 유전자 변형에 의한 품종 개량이 달성될 수 있다는 것을 보여주며, 질병 저항성 돼지 생산을 위한 육종 시작점을 제공한다. 종간 배반포 보완(interspecies blastocyst complementation)은 이종 만능 줄기세포 유도체(xenogenic pluripotent stem cell derivatives)의 장기 특이적 생산(organ-specific enrichment)을 가능하게 한다. CRISPR-Cas9 매개 접합자 유전자 편집(CRISPR-Cas9-mediated zygote gene editing)을 이용하여 췌장 생성(pancreatogenesis), 신장 생성(nephrogenesis), 간 생성(hepatogenesis) 및 혈관 생성(vasculogenesis)이 불가능 생쥐 숙주를 만들었으며, 이러한 숙주와 배반포 보완 플랫폼을 결합하여 키메라를 만들었다. 또한 돼지와 소 같은 유제류(ungulate)의 섬유아세포(fibroblasts)를 이용하여 CRISPR-Cas9 매개 유전자 편집과 체세포 핵 치환(somatic cell nuclear transfer) 과정을 거쳐 복제 배아(genome-edited cloned embryos) 를 생산하였다. 복제 배아의 1차 배양 섬유아세포(primary cultured fibroblasts)를 재복제하여 배반포 보완을 위한 숙주 배아로 이용하였다. CRISPR-Cas9 유전자 편집 기술과 종간 배반포 보완 플랫폼 전략의 조합은 유전자 변형 돼지를 생산하는 데 유용하다. 본 논문에서는 CRISPR/Cas9 유전자 편집 기술과 배반포 보완 플랫폼, 질병 저항성(disease resistance) 돼지, 이종장기이식(xenotransplantation) 목적의 키메라 생산을 소개하고자 한다.

최근 급속하게 발전하고 있는 유전자교정기술은 식물이 생산하는 특정 이차 대사산물의 집적을 유도하기 위한 식물대사공학연구에 아주 유용하게 이용되고 있다. 특히 이들 기술 들을 이용하여 만든 유전자교정 작물 중에 일부는 외래 DNA 단편이 잔존 하지 않기 때문에 기존의 유전자변형작물의 안전 관리규정에 적용되지 않을 수 있다는 장점이 있다. 따라서 본 리뷰는 phenylpropanoid 대사과정에 의하여 합성되는 다양한 종류의 이차대사산물을 집적시키기 위한 유전자교정 기술의 적용 연구결과 들을 조사하였다. 먼저, phenylpropanoid 생합성 대사과정에 관여하는 다양한 효소를 암호하는 유전자들을 목표로 하여 식물의 종에 따라 특이하게 집적되는 flavonoids, anthocyanin, 수용성 tannins, 로즈마린산 등의 집적을 유도하거나 화색을 변경하는 등의 성공적인 연구결과들을 검토하였다. 또한, phenylpropanoid 대사과정의 조절에 최종조절 스위치 역할을 하는 수많은 종류의 MYB 전사인자를 암호 하는 유전자를 목표로 하여 CRISPR 유전자교정을 시도한 연구결과들로부터 식물의 이차세포벽 형성에 관여하는 lignin, 물관부, cellulose 등의 생합성 조절 기작을 이해하고 MYB family에 속하는 수많은 종류의 유전자에 대한 개별적인 기능 분석 연구결과들을 조사 분석하여, 문제점 및 향후 연구 방향 등을 검토하였다.

Porcine Reproductive and Respiratory Syndrome (PRRS) is the most economically important disease in swine in North America, Europe and Asia. PRRS is caused via infection of the pulmonary alveolar macrophages (PAMs) with the PRRS virus (PRRSV) causing respiratory illness and high fever in young growing pigs that predisposes them to secondary bacterial infections. PRRSV also causes severe reproductive failure in sows and boars. Although research is ongoing, PRRSV continues to elude a successful vaccine. In 2014, piglets were born with a gene edit in exon 7 of the Cluster of differentiation 163 (CD163) gene introduced by using the CRISPR/Cas9 site-directed nucleases system. The resulting litters of pigs were either challenged with multiple PRRSV isolates at 3 weeks of age or bred at maturity for a challenge with pregnant sows. The challenges demonstrated that the pigs were completely resistant to infectivity to both Type 1 and 2 isolates as measured by clinical signs, viremia, antibody response and lung histopathology. In a follow-up study, pregnant CD163-/- pigs were also challenged with PRRSV to determine if absence of CD163 in the dam should be sufficient to protect the CD163+/- fetuses that have functional CD163 protein. The wild-type sow and fetuses were actively infected with the PRRSV and one sow aborted. The CD163-/- sows carrying both the CD163-/- and CD163+/- fetuses were all negative for PRRSV nucleic acid and showed no sign of fetal or placental failure. The results of this study clearly demonstrate that the absence of CD163 in the sow is sufficient to protect a PRRSV-susceptible CD163+/- fetus. Gene editing of CD163 in pigs, via CRISPR/Cas9, successfully blocked PRRSV infectivity in young growing pigs and pregnant sows and their fetuses. This is a great example of the potential of utilizing gene editing to improve animal agriculture.

CRISPR/Cas9-induced knock-out/-in can be occurred at specific locus in the genome by non-homologous end joining (NHEJ) or homology directed repair (HDR). Here, we demonstrate the targeted insertion into the specific loci of embryo fertilized by semen from transgenic cattle via CRISPR/Cas9 system. Recently, we published on the efficient generation of transgenic cattle using the DNA transposon system (Yum et al. Sci Rep. 2016 Jun 21;6:27185). In the study, eight transgenic cattle were born following transposon-mediated gene delivery system (Sleeping Beauty and Piggybac transposon system) via microinjection. In the analysis of their genome stability using next-generation sequencing, there was no significant difference in the number of genetic variants between transgenic and non-transgenic cattle. All the transgenic cattle have grown up to date (the oldest age: 33 months old, the youngest age: 15 months old) without any health issue. One of transgenic male cattle expressing GFP reached puberty and semen was collected. Over 200 frozen semen straws were produced and some were used for in vitro fertilization (IVF). On seven days after IVF, expression of GFP was observed at blastocyst stage and was seen in 80% of the embryos. Another application is to edit the GFP locus of the transgenic cattle because long-term and ubiquitous expression of transgene didn’t affect their health. In one cell stage embryos produced using GFP frozen-thawed semen, microinjection of sgRNA for GFP, Cas9, together with donor DNA that included RFP and homology arms to link the double-strand break of sgRNA target site into fertilized eggs resulted in expression of RFP. This indicated that the GFP locus of transgenic cattle shows potential candidates for stable insertion of the functional transgene. Knock-out/-in for editing GFP locus using CRISPR-Cas9 might be a valuable approach for the next generation of transgenic models by microinjection. In conclusion, we demonstrated P-112 that transgenic cattle via transposon system are healthy to date and germ-line competence was confirmed. The GFP locus will be used as the potential target site for future gene engineering via genome-editing technology. Finally, all those animals could be a valuable agricultural and veterinary science resource for studying the effects of gene manipulation on biomedical research and medicine. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550-20160004).

The CRISPR/Cas9 system is proved to be a powerful tool for knock-out and knock-in in various species. By introducing genetic materials of two components (Cas9 and small guide (sg) RNA) into cells or pronuclear of the fertilized embryo, gene editing occurs. Some studies reported that efficiency of gene editing would be increased as Cas9 was integrated into cells or animals since Cas9 is indispensable in the CRISPR/Cas9 system. Accordingly, the production of Cas9 expressing cattle may provide the broadly used gene editing platform in cattle. For this study, Cas9 and RFP genes were cloned into PiggyBac (PB) transposon system. PB-Cas9-RFP and transposase were microinjected into 1436 in vitro fertilized embryos and 241 blastocysts were formed. Blastocysts with RFP expression accounting for 14.1% of total formed blastocysts were selected and transferred into 5 recipient cow. After gestation periods, four transgenic cattle were delivered without any veterinary assistance. From a transgenic cattle, ear skin tissue was collected for primary culture. On those primary cells, sgRNAs in DNA form for various genes such as PRNP, RB1 and BLG were transfected as 2ug of sgRNA per 5x105 cells using Nucleo factor system (Neon®, invitrogen, program#16). As expected, every group of each sgRNA delivered was confirmed to be mutated by T7E1assay. Those data demonstrated that for the first time, transgenic cattle with Cas9 expression were born, grown up to date and will be avaluable resource for genome-editing in cattle. This work was supported by BK21PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU550-20160004).

With the development of the third-generation gene scissors, CRISPR-Cas9, concerns are being raised about ethical and social repercussions of the new gene-editing technology. In this situation, this article explores the legislation and interpretation of the positive laws in South Korea. The BioAct does not specify and regulate 'gene editing' itself. However, assuming that genetic editing is used in the process of research and treatment, we can look to the specific details of the regulations for research on humans as well as gene therapy research in order to see how genetic editing is regulated under the Bio- Act. BioAct differentiates the regulation between (born) humans and embryos etc. and the regulation differ entirely in the manner and scope. Moreover, due to the fact that gene therapy products are regarded as drugs, they fall under different regulations. The Korean Pharmacopoeia Act put stringent sanctions on clinical trials for gene therapy products and the official Notification "Approval and Examination Regulations for Biological Products, etc." by Food and Drug Safety Administration may be applied to gene editing for gene therapy purposes.

Zinc finger nucleases (ZFNs) have been used for targeted mutagenesis in eukaryotic cells. Custom-designed ZFNs can induce double-strand breaks (DSBs) at a specific locus. Our custom ZFN dimer was designed 3-finger of left and 4-finger of right with 2 kb size using 2A. A Ti-plasmid vector, pTA7002 containing the target site of SSS4A gene for a ZFN pair, that was shown to be active in yeast, was integrated in the rice genome. This promising technique for genome engineering was induced into 4 exon region of SSS4A gene in rice genome using Agrobacterium-mediated transformation. The SSS4A full-length cDNA was 5,070 bp consisting of a 318 bp 5′-untranslated region (UTR), a complete ORF of 2,928 bp encoding a polypeptide of 975 amino acids and a 3′-UTR of 1,824 bp. The vector is based on glucocorticoid receptor inducible gene expression system. Thus, SSS4A::ZFN expression was tightly controlled and the phenotype in low concentrations 10uM of the glucocorticoid hormone dexamethasone (DEX). In plant cells, transient ZFN expression is achieved by direct gene transfer into the target cells. For an alternative, ZFN delivery and production of mutant plants using a tobacco transient expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of plants. ZFN activity was determined by PCR and sequence analysis of the target site. ZFN induced plants were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1and 100 bp and insertions ranging between 1 and 10 bp. Our results describe an alternative to direct gene transfer for ZFN delivery and for the production of mutated rice.