축산업의 유전자공학 기반 고도화 기술: 우수한 소 형질 개량을 위한 다양한 전략

학위논문(박사) -- 서울대학교대학원 : 수의과대학 수의학과, 2023. 2. 장구.This study aimed to produce specific gene mutated (PRNP and MSTN) cattle through cytoplasmic microinjection based on the CRISPR/Cas9 system and to verify that mutant traits are transferred to the next generation by germline transmission. First, to produce PRNP-mutated cattle, piggyBac transposon and CRISPR/Cas9 were used. A transposon vector with Cas9, GFP, and sgRNA for PRNP was applied to bovine somatic cells and embryos. Cas9 and sgRNA were inserted into the bovine genome and PRNP mutation was induced. Then, GFP-expressing blastocysts were selected and transferred into 18 surrogates. Finally, 7 calves were successfully born. Among them, 6 calves (#P1, #P3, #P4, #P5, #P6, and #P7) showed vector insertion (Cas9, sgRNA for PRNP), and their mutation rates were 4.1%, 48.3%, 0.2%, 0.0%, 99.6%, and 94.4%, respectively. However, GFP expression and Cas9 activity were observed in only 4 calves (#P1, #P3, #P4, and #P7). To verify germline transmission, #P3 and #P7 germ cells were cultured in vitro, and PRNP mutation was detected in their blastocysts. As further gene editing, GGTA1 mutation was introduced into the embryos using electroporation. Using germ cells (#P3 and #P7), 7 F1 calves became pregnant. In F1 cattle, the gene of interest in All-in-one DNAs (Cas9, GFP, and sgRNA for PRNP) was identified, and PRNP mutation was detected. In addition, to study PRNP function in detail, conditional PRNP-mutant cattle were produced based on the Cre/loxP system. After Cre treatment, the somatic cells of the cattle expressed Cas9, but showed no PRNP mutation. As a result of germline transmission of the conditional PRNP male cattle, transgene integration and GFP expression were observed in blastocysts fertilized with semen. Second, to generate MSTN-mutant cattle, cytoplasmic microinjection based on the CRISPR/Cas9 system was used. Through this, MSTN-mutant calves were successfully produced. The MSTN mutation pattern was the same with 12-base pair deletion, and, in case of calf #17, enhanced muscle growth was observed. Furthermore, blood analysis results showed no abnormalities in the MSTN-mutant cattle. Next, whether MSTN mutation was transferred to the next generation (F1) was confirmed. For this purpose, oocytes and semen were collected after sexual maturation of the MSTN cattle (#6 and #17), and embryos produced by in vitro fertilization were analyzed. In addition, the embryos were subjected to additional gene (PRNP) editing using electroporation. Embryos produced by in vitro fertilization with the MSTN male and female cattle were transferred to a surrogate, and 1 calf was successfully born. MSTN heterozygous mutation was observed on sequencing of the F1 calf, which had no health issues. As a further experiment, using electroporation, the additional gene-edited embryos fertilized with the MSTN male sperm showed high PRNP mutation rate (86.2 ± 3.4%). In conclusion, this study is the first to produce PRNP-mutant cattle using transposon and the CRISPR/Cas9 system and MSTN-mutant cattle without exogenous gene integration. In addition, germline transmission was confirmed. The CRISPR/Cas9 system can be used to produce specific gene-mutant cattle with high efficiency and can be applied in various fields, such as livestock industry and veterinary medicine.본 연구의 목적은 CRISPR/Cas9 이용하여 특정 유전자(PRNP, MSTN)을 타겟팅하여 돌연변이가 유도된 형질전환 소를 생산하는 것과 그 돌연변이가 다음 세대로 정상적으로 생식선 전이가 일어나는 것을 확인하는 것이다. 첫번째 PRNP 돌연변이 소를 생산하기 위해서 microinjection과 Piggybac 트랜스포존을 이용하여, PRNP 유전자에 돌연변이를 유도하였다. Cas9, GFP, PRNP 가이드 RNA가 포함된 트랜스포존 벡터가 소의 체세포와 수정란의 유전체에 정상적으로 삽입이 되고, PRNP 유전자에 돌연변이가 발생되는 것을 확인되었다. GFP 발현하는 배반포를 선별하여 18마리의 수란우에 이식하여 7마리의 송아지를 생산하였다. 생산된 7마리 중에서 4마리에서 성공적으로 PRNP 돌연변이를 보여주었다. 생식선 전이를 확인하기 위해서 #P3, #P7 소의 정상적인 성 성숙 이후 생식세포를 이용하였다. 이들의 생식세포를 이용하여 생성된 배반포에서 PRNP 돌연변이를 확인되었고, 수정란 이식을 통해서 성공적으로 F1 송아지에서 PRNP 돌연변이가 정상적으로 생식선 전이가 이루어지는 것을 관찰하였다. PRNP 유전자를 구체적으로 분석하기 위해서, Cre/loxP 시스템을 기반으로 하여, conditional PRNP 돌연변이 소를 생산하였다. 하지만, F0의 체세포에서 Cre 단백질 처리이후 Cas9 단백질 발현은 정상적으로 이루어 졌지만, PRNP 유전자 돌연변이는 발생되지 않았다. 하지만, conditional PRNP 돌연변이 수컷에서 정상적인 생식전전이를 수정란 체외배양으로 생산된 배반포에서 관찰이 되었다. 두번째 연구로서, 외래 유전자 삽입이 없는 MSTN 돌연변이 소를 생산하기 위해서 Cas9 mRNA와 sgRNA for MSTN을 수정란에 세포질 microinjection 하였다. 생산된 배반포는 26마리에 이식을 진행하고, 17마리의 송아지를 얻었다. 그 중 3마리에서 MSTN 돌연변이가 관찰이 되었다. 태어난 MSTN 돌연변이 소에서 off-targeting 영향과 혈액검사 결과 건강상 문제가 없음이 확인되었다. 다음으로, #6, #17 MSTN 돌연변이가 생식선 전이가 되는지를 수정란 수준에서 확인하였다. #6과 #17의 체외수정으로 생산된 배반포를 수란우에 이식을 하여 성공적으로 F1 송아지를 생산하였다. 태어난 송아지에서는 MSTN 돌연변이를 보여주었으며, 건강상에 문제는 관찰되지 않았다. 본 연구는 최초로 CRISPR/Cas9 기반으로 하여 PRNP 돌연변이 소와 외부 유전자가 삽입되지 않은 MSTN 돌연변이 소를 성공적으로 생산하였다. 또한 생식선 전이를 통해 이들의 돌연변이가 다음 세대로 돌연변이가 전달되는 것을 확인하였다. 이러한 연구 결과는 CRISPR/Cas9 시스템을 이용하여 특정 유전자 돌연변이 소를 높은 효율로 생산할 수 있음을 보여주었으며, 축산업 및 수의학 등의 다양한 분야에서 적용될 수 있을 것이다.ABSTRACT 3 TABLE OF CONTENTS 6 LIST OF TABLES 9 LIST OF FIGURES 11 LIST OF ABBREVIATIONS 14 PUBLICATION LISTS 17 PART I. LITERATURE REVIEW 23 1. Production of transgenic cattle 24 2. Gene engineering tools 33 3. PiggyBac transposon 52 PART II. GENERAL METHODOLOGY 63 1. Reagents 64 2. Primary cell culture 64 3. In vitro maturation 64 4. In vitro fertilization and in vitro culture of embryo 65 PART III. GERNERATION OF GENETICALLY ENGINEERED CATTLE 67 Chapter Ⅰ. The production of PRNP gene mutated cattle by CRISPR/Cas9 and piggyBac transposon 68 1. Abstract 69 2. Introduction 71 3. Materials and methods 74 4. Results 88 5. Discussion 125 Chapter II. The production of MSTN gene mutated cattle by CRISPR/Cas9 129 1. Abstract 130 2. Introduction 131 3. Materials and methods 134 4. Results 144 5. Discussion 157 Chapter III. Stable germline transmission from the MSTN gene mutated cattle 162 1. Abstract 163 2. Introduction 165 3. Materials and methods 168 4. Results 176 5. Discussion 190 REFERENCES 197 국문 초록 225박

Developing generic and modular approaches to targeted cancer cell therapeutics

The intricacies and complexities of cancer render it a difficult disease to treat, and existing treatments are frequently non-selective. This project investigated two selective cancer therapeutic approaches: organelle-targeted drug delivery, and genetic re-wiring to achieve a switchable CAR-T model. Cancer mitochondria are different to healthy cells, including a higher mitochondrial membrane potential (MMP) which can be exploited for selective delivery of a therapeutic. Cyanine dyes Cy3 and Cy5, along with a dimer Cy3- Cy5 and a CPP conjugate Cy3-Cy5-R8 were characterised, and all constructs stained the mitochondria of HeLa in an MMP-dependent manner. Staining capacities of Cy3, Cy5 and Cy3-Cy5 were not hindered by serum proteins or endocytosis inhibition. Conversely, serum proteins reduced Cy3-Cy5-R8 staining capacities, and its uptake was endocytosis-dependent. Cy3 was subsequently tested as a mitochondrial-drug delivery vehicle, and Cy3 conjugation to mitochondrial toxins improved EC50 values by up to 1000-fold. The Cy3-drug conjugates were more toxic to cancerous (HeLa) vs noncancerous cells (HEK293), but toxicity was still present in HEK293. Further studies are therefore needed to enhance Cy3-drug selectivity to cancer cells. Cancers can alternatively be targeted via CAR-T cell immunotherapy; however, CAR-T frequently over-activate and bring unwanted toxicity to the patient. Through genetic code expansion, a new logic gates approach was developed. 11 quadruplet-decoding pyrrolysyl tRNA variants that incorporate BocK were analysed. Unlike their literature representation in E. coli, only five variants were functional in HEK293. Increasing tRNA copy number from 1 to 4 improved BocK-incorporation, and PylRS/tRNA was found to function orthogonally alongside a mutant TyrRS/tRNA pair. A split GFP reporter system was subsequently developed, where AND and OR logic operations were successfully generated whereby GFP output can be controlled via the unnatural amino acid makeup of the cellular media. The cell circuits developed here provide a new approach to mammalian cell logic operations, and can potentially be translated into a switchable ON/OFF CAR-T model