CRISPR Based Disease Mouse Model
CRISPR 기반의 질환 모델 마우스

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dc.description학위논문 (석사)-- 서울대학교 대학원 : 국제농업기술대학원 국제농업기술학과, 2018. 2. 염수청.-
dc.description.abstractCRISPR/Cas system has been a powerful tool for genome engineering which led to tremendous development in biological study. CRISPR/Cas Type Ⅱ inter alia, Streptococcus pyogene Cas9 (SpCas9) was the first and widely used orthologue because it shows high cleavage efficiency of double strand DNA (dsDNA) and low off-target effect. Recently, novel orthologue campylobacter jejuni Cas9 (CjCas9) was reported as the smallest Cas9. Discovering novel orthologue is drawing attention because this doesnt just stop on finding new type of CRISPR it gives prospects for various application such as disease animal modeling and gene therapy. In addition, ultimate goal for overall applications is to cure genetic disease in human. Therefore, as the research advances high cleavage efficiency as well as low off-targets are being emphasized for CRISPR system. So in this study I focused on testing overall efficacy of SpCas9 and CjCas9 through animal modeling.
Firstly, by using classical orthologue SpCas9, I targeted Hr gene to generate hairless mouse. With using 4 sgRNAs in exon 3, cleavage activity revealed 30% (3/10) in blastocyst and 20-28.5% (1/5-2/7) in pup. Among the mutant pups, #1-4 mice (-3 and -34 amino acid deletion) and #2-4 mice (premature termination codon(PTC) and -50 amino acid deletion in genomic locus) were germline transmitted to observe phenotype. In #2-4 mice line, hairless phenotype was appeared about 2week postpartum whereas #1-4 mice line didnt showed the hair loss. This study verified SpCas9 cleavage activity and in addition it suggests appropriate model for dermatological research and in vivo imaging.
During our study novel orthologue CjCas9, briefly stated above, was reported. However previous studies were conducted in in vitro and overall efficacy were not established. Albeit several opinions of cleavage ability about CjCas9, I investigated cleavage efficiency as well as off-target possibilities in in vivo. Totally, in zygote and generated mice cleavage efficiency showed 38-100% which showed unexpected high rate. Besides off-target analysis showed ≤1.6% which indicates high specificity. In addition, most recent report related to off-target analysis using bioinformatics study conducted by Schaefer et al demonstrated high off-targets in SpCas9. However, on the line of our study with CjCas9 I found this was a misleading interpretation due to aligning SpCas9 mediated mutant mice sequence to wrong control mice sequence. Consequently, CjCas9 showed high cleavage efficiency as well as high specificity and this suggests safe gene targeting which can broaden biological development.
With our discovery of efficiency and accuracy in CjCas9, I applied it to multiple gene related pancreatic cancer modeling with in vivo gene targeting mediated by adeno-associated virus (AAV). In this study, 5 genes (Kras, Ink4a, Tp53, Smad4, Brca2) related to pancreatic cancer were selected and SpCas9 and CjCas9 were directly compared by targeting same genes. Cleavage rate were confirmed in zygote and it revealed similar rate between SpCas9 and CjCas9. Then, in vivo gene targeting in adult mice was proceeded by directly injecting AAV packaged molecule into pancreas common bile duct. After surgery monitoring were conducted every week and since cancer formation takes at least 9month post-surgery, this studies are ongoing.
In conclusion, I demonstrate safe and stable ability in CjCas9 as well as SpCas9 by generating animal model in various way. This suggest prospects for broad application.
dc.description.tableofcontentsChapter I: Generation of Hairless mouse using CRISPR/Cas9 system 1
1. Introduction 2
2. Material and method 4
2.1. Animal 4
2.2. Preparation of Cas9 mRNA and sgRNA 4
2.3. Microinjection into embryos 7
2.4. Genotyping and sequencing 7
2.5. Germline transmission 9
3. Results 10
3.1. sgRNA efficiency test in C57BL/6 embryos 10
3.2. Generation of hairless mouse by CRISPR/Cas9 12
3.3. Germline transmission 16
3.4. Hairless Phenotype 19
4. Discussion 20
Chapter 2: Whole Genome Sequencing of Genome Modified mice generated with Campylobacter jejuni Cas9 reveals High Specificity of CRISPR/Cas9 23
1. Introduction 24
2. Material and method 27
2.1. Animal 27
2.2. Preparation of Cas9 mRNA and sgRNA 27
2.3. Embryo microinjection 30
2.4. Genotyping and sequencing 30
2.5. In silico design of Off-target sites 32
2.6. Targeted deep sequencing 32
2.7. Whole genome sequencing 34
3. Results 35
3.1. High cleavage activity of CjCas9 in knock out mice generation 35
3.2. Off-target Analysis showed high specificity 42
3.3. WGS based bioinformatics analysis revealed different off-target rate by the control mice 44
4. Discussion 48

Chapter 3: In vivo Multiple Gene Targeting with Campylobacter jejuni cas9 for Pancreatic Cancer
1. Introduction 52
2. Material and method 54
2.1. Target gene sgRNAs preparation 54
2.2. Generation of SpCas9 overexpression mice 56
2.3. Vector Preparation of AAV2-SpCas9 and AAV2-3sgRNAs 56
2.4. AAV preparation 59
2.5. AAV injection into pancreas 59
3. Results 62
3.1. SgRNA cleavage activity test of SpCas9 and CjCas9 in C57BL/6 embryo 62
3.2. SpCas9 expression was confirmed by RFP signal in pancreas 68
3.3. In vivo Pancreas-tropism of AAV2 70
3.4. Monitoring 73
4. Discussion 74

References 78
국문초록 84
dc.format.extent2549377 bytes-
dc.publisher서울대학교 대학원-
dc.subjectAnimal model-
dc.subjectCampylobacter jejuni-
dc.subjectin vivo gene targeting-
dc.subjectPancreatic cancer-
dc.titleCRISPR Based Disease Mouse Model-
dc.titleStudy for Genome Editing Efficiency of Campylobacter Jejuni Cas9 and In Vivo Gene Targeting for Pancreatic Cancer-
dc.title.alternativeCRISPR 기반의 질환 모델 마우스-
dc.title.alternative캠필로박터 제주니의 유전자 교정 효율 분석 및 생체 내 유전자 적중을 통한 췌장암 모델 확립연구-
dc.contributor.AlternativeAuthorYoo Jin Chang-
dc.contributor.affiliation국제농업기술대학원 국제농업기술학과-
Appears in Collections:
Graduate School of International Agricultural Technology (국제농업기술대학원)Dept. of International Agricultural Technology (국제농업기술학과)Theses (Master's Degree_국제농업기술학과)
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