최근 전세계적으로 기능성 성분이 강화된 유전자교정 작 물의 생산 및 소비가 증가하고 있다. 하지만 유전자교정 작물의 유전독성에 관한 소비자의 우려도 증가하고 있어 과학적인 자료 확보 및 정보 공유에 대한 인프라가 필요한 실정이다. 본 연구에서는 CRISPR/Cas9 시스템을 활용한 유 전자교정 토마토 동결건조물(LTT)이 DNA나 염색체에 직접 적인 손상을 일으키고 형태적 또는 기능적 이상을 유발하 는지 여부를 확인하기 위해 유전독성 평가를 수행하였다. 이를 위하여 미생물 복귀돌연변이시험, 염색체이상시험, 골수세포를 이용한 체내 소핵시험을 국제적으로 표준화된 OECD Guidelines에 따라 시험을 진행하였다. 복귀돌연변이 시험에서 LTT는 S9의 존재 여부와 관계없이 Salmonella typhimurium 균주 TA98, TA100, TA1535 및 TA1537, 그리 고 Escherichia coli WP2 uvrA에서 복귀돌연변이를 유발하 지 않았다. LTT는 CHL 세포의 수적이상 중기상과 구조적 이상 중기상 등의 염색체 이상을 유발하지 않았다. 또한, LTT는 다염성 적혈구에서 소핵화된 다염성 적혈구의 빈도 를 증가시키지 않았다. 이러한 연구를 통해 CRISPR/Cas9 시스템을 활용한 유전자교정 토마토의 안전성을 검증하고, 향후 CRISPR/Cas9 시스템을 활용한 유전자교정 작물의 유 전독성을 평가하는 기초 자료로 사용될 수 있을 것이다.

배스(Micropterus salmoides)는 수생태계에서 최상위단계에 위치하는 생태계교란 어종으로 심각한 담수생태계의 불균형을 초래하고 있다. 배스의 퇴치 및 관리를 위한 다양한 시도를 하고 있지만 효과적인 방안은 없는 상황이므로 배스의 고유한 특성에 기반한 개체군 감소의 효율성을 극대화할 수 있는 방식을 모색하였다. 본 연구에서는 배스의 Transcriptom 분석으로 Unigene contigs는 182,887개, 그리고 정자-난자 인식 단백질인 IZUMO1과 Zona pellucida sperm-binding protein의 유전자에서 CRISPR/Cas9 system을 적용할 최종 Target sequence는 12종을 산출하였다. 각 Target sequence를 인식할 수 있는 12종의 sgRNA를 합성한 후 후속 연구에 사용할 12종의 Cas9-sgRNA ribonucleoprotein (RNP) complex를 제작하였다. 본 연구에서는 차세대염기서열 분석법으로 정자-난자 인식 단백질을 암호화하는 유전자를 탐색하였고, CRISPR/Cas9 system으로 유전자를 편집하여 번식행동은 하지만 수정란을 형성하지 못하는 생식세포를 생산하는 불임개체를 유도하기 위한 조성물 개발 과정을 확립하였다. 그리고 배스와 동일한 수계에 있는 고유 생물종의 서식에는 영향을 미치지 않는 생태교란종 관리 방안으로서의 유용성을 검증하기 위한 후속 연구의 귀중한 기초 자료를 확보하는데 기여했다고 판단된다.

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.

Prostaglandin E2(PGE2) is an autocrine and paracrine signal in insects and other animals. Its signal pathways in target cells are well understood in mammalian system but not in insects. Here, we assessed PGE2 signaling in hemocytes of Spodoptera exigua through knocking-down of signal component genes by RNA interference (RNAi) and knocking-out (KO) of PGE2 receptor using CRISPR-Cas9. From S. exigua transcriptome, we selected hemocyte signaling components and analyzed their functions in cellular immune responses through RNAi. KO mutant against PGE2 receptor exhibited severely hampered larval development and adult fecundity.

Insulin/IGF signaling (IIS) regulates multiple physiological processes such as larval growth, reproduction, and life span in many organisms including legume pod borer, Maruca vitrata (Lepidoptera: Crambidae). RNA interference of IIS components, insulin receptor (InR) and Forkhead Box O (FOXO), impaired larval growth and female reproduction. To further validate the physiological roles of InR and FOXO, we generated knock-out (KO) mutants using CRISPR/Cas9-mediated genome-editing technology. Both KO mutants exhibited delayed larval growth and reduced pupal and adult body sizes. In conclusion, these results demonstrated the critical role of insulin signaling (IIS) pathway to control M. vitrata growth and development.

A specific serotonin receptor (Se-5HTR) has been identified in the beet armyworm, Spodoptera exigua and classified into 5-HT7 type. Se-5HTR expression was up-regulated in hemocytes and fat body in response to immune challenge. As being a GPCR, this receptor is presumably coupled with intracellular trimeric Gαs protein activating cAMP-dependent protein kinase (PKA) pathway to regulate several cellular functions. RNA interference (RNAi) of Se-5HTR as well as its downstream signal proteins exhibited significant suppression in cellular immune responses including nodulation and phagocytosis. Application of inhibitors to the signaling cascade suppressed the immune responses as well. To validate the Se-5HTR involvement in mediating cellular immunity, 5-HTR knock-out mutants were developed using CRISPR-Cas9 technique and suffered significant developmental anomalies.

Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of α-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

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 clustered regularly interspaced short plalindromic repeats(CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. We applied CRISPR/Cas9 system to generate hG-CSF targeted pig parthenogenetic embryos. Using sigle guided RNA targeted to pig hG-CSF genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. The CRISPR/Cas9 vector were diluted in Tris-EDTA buffer (TE buffer) and injected with different concentration of 0 (sham injection), 2.5 and 25 ng/ul. In results, regardless of the concentrations of vector, the cleavage and blastocyst rate were not significantly different among three groups. Since plasmid DNA was used for microinjection, we investigated whether DNA vectors were integrated into the genome. Genomic PCR of the coding sequence of Cas9 variants and hG-CSF was performed to detect genomic integrants. Each blastocysts were collected into a microtube, and then PCR was performed. Overall 32 embryos are not expressed targeted gene.

CRISPRs(clustered regularly interspaced short palindromic repeats) / CRISPR - associated(CAS) system has been used genome editing technology. Genome stage modification using CRISPR/CAS9 system can be used to wide research for the gene functional study and therapeutics. However, improving of CRISPR/CAS9 system in efficiency is essential for application in various fields. Here, we treated various chemicals during the procine early embryo development to increase the mutation of target site by NHEJ(non-homologous end joining). Firstly, we confirmed the chemical toxicity after parthenogenetic activation and then check embryo puality using by counting of total cell number and TUNEL Assay in blastocyst satge. To check any improvement on mutation rate by NHEJ pathway. AZT(3′-Azido-3′-deoxythymidine, antiretroviral drug – 0.1 μM) was treated after injection of cas9 and sgRNA target to OCT4 exon 5 during the zygote stage, followed by PCR sequencing. As a result, AZT treated group shows a significantly increased in knock-out efficiency as a consequence of NHEJ. Nocodazole(anti-neoplastic agent – 200ng/ml), RO-3306 (specific inhibitor of CDK1 - 10 μM) and NU-7026(PKC signalling inhibitor - 50 μM) was treated after injection of cas9 and sgRNA with eGFP vector during the zygote stage(hpa8~hpa20) and checked a efficiency of knock-in by PCR sequencing. Interestingly, nocodazole treatment groups increased of insertion of eGFP sequence in blastocyst stage compared with non-treat group(control : 8.33%, nocodazole treatment : 16.67%). However, RO-3306 and NU-7026 made a no impact. In summary, CRISPR/CAS9 system with treatment of chemicals during porcine embryogenesis can be improving of site-specific mutation and enhancement of CRISPR genome editing.

KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.