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.

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).

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.

The formation of definitive endoderm (DE) is a fundamental step for the development of the gastrointestinal tract, respiratory tract and endocrine organs. We present a CRISPR-based pooled screening approach to identify genes which contribute to DE induction from hiPSCs in vitro. CRISPR-based pooled genetic screens in mammalian cell culture enable researchers to identify genes required for a cellular phenotype of interest in an unbiased way. To enable a CRISPR-based forward genetic screen for identifying regulatory genes required for DE differentiation from hiPSCs, we performed pooled screens using a human genome-scale CRISPR knockout library. In addition, we performed a transcriptional activation screen using a lentiviral CRISPRa library to identify the downstream targets of the TGF/nodal/activin signaling pathway, which is a key signaling pathway for DE specification. We identified several signaling pathways including TGFβ, Erk, JNK, and CREB pathways are involved with DE differentiation. We suggest that CRISPR-based pooled genetic screens are a useful tool to identify key signaling pathways and genes required for in vitro differentiation processes and are served as a platform to improve differentiation protocols.

유전자 가위(Engineered nuclease)는 최근 유전자의 특정염기서열을 인식하여 목적 유전자 부위만을 정확히 편집하여 형질 교정을 유도하는 획기적인 기술이다. 본 연구에서는 세포벽으로 인해 형질교정율이 동물 시스템에 비하여 상대적으로 낮은 식물세포에 적용시켜 효율을 높이기 위한 조건을 확립하고자 함을 연구목적으로 하였다. 타겟 유전자인 질소환원효소(Nitrate reductase)에 맞춤 제작된 3세대 유전자가위 RGEN (RNA-guided Engineered nuclease)을 이용하여 페튜니아의 원형질체 수준에서 고효율의 형질교정을 유도시키는 조건을 조사하였다. 종자로부터 기내에서 자란 폐튜니아의 어린 잎을 사용하여 cellulose, viscozyme, pectinEX이 포함된 혼합 효소액을 처리한 후 원형질체의 분리를 유도하였다. 예비 실험으로 PEG와 형질전환에 사용된 플라스미드 DNA인 pBI1221-GFP의 농도를 조절하여 원형질체에 도입한 결과, PEG의 농도가 40%이고 Plasmid DNA의 농도를 50ug을 이용하였을 때, 30% 이상의 가장 높은 유전자 도입 효율을 보이는 것을 확인하였다. 동일한 조건으로 페튜니아 NR 유전자에 맞춤 제작된 CRISPR/Cas9을 원형질체에 도입하여 세포배양을 실시한 후 배양세포로부터 DNA를 추출하여 mid-seq을 통한 변이체 발생 비율을 확인한 결과 최대 12%까지 타겟 유전자의 교정이 유도됨을 확인할 수 있었다. 본 연구에서 확립한 조건을 바탕으로 다른 가지과 작물의 다양한 선별 유전자에 적용시켜 목적 형질의 교정을 유도할 수 있는 새로운 작물 육종기술로 본 유전체 편집기술이 이용되도록 그 기반을 확립할 것이다.