Browse

Highly specific genome editing in human cells and plants using CRISPR systems

DC Field Value Language
dc.contributor.advisor이형호-
dc.contributor.author김정은-
dc.date.accessioned2017-07-14T05:58:58Z-
dc.date.available2017-07-14T05:58:58Z-
dc.date.issued2017-02-
dc.identifier.other000000140928-
dc.identifier.urihttps://hdl.handle.net/10371/125337-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 화학부, 2017. 2. 이형호.-
dc.description.abstractTargeted genome engineering with RNA-guided engineered nucleases (RGENs) repurposed from the clustered regularly interspaced short palindromic repeat (CRISPR) systems has recently received significant attention because of their simple design and preparation. Although, RGENs-mediated genome editing has been successfully performed in cultured cells and model organisms, developing a highly efficient and specific CRISPR-based genome editing platform is still a major challenge in this area.
In this study, I develop and optimize the new CRISPR-based genome editing platforms and investigate their target specificities. First, I present delivery of RGEN RNPs in plant cells. RGEN RNPs enabled DNA-free genome editing in plant cells at high frequencies up to 44%. Whole plants were successfully regenerated from gene-edited cells and no apparent off-target mutations were detected in these mutants. This method may reduce the likelihood of DNA insertion and potential off-target effects and alleviate the concerns related to genetically modified plants. Second, I optimize the Cpf1-mediated genome editing system and investigate its target specificity. CRISPR-Cpf1 system induced targeted genome modification efficiently in human cells. Mismatched crRNAs test and genome-wide off-target analysis showed that this nuclease yielded DNA cleavages at the target site in a highly specific manner. These results indicate that CRISPR-Cpf1 is suitable for precision genome editing and will be a good option for RNA-guided genome editing.
-
dc.description.tableofcontentsCAHPTER 1. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins 1

INTRODUCTION 2

MATERIALS AND METHODS 6
1. Cas9 protein and guide RNA preparation 6
2. Construction of RGEN-encoding plasmids 6
3. Protoplast culture and isolation 7
4. Protoplast transfection 8
5. Genomic DNA isolation 9
6. T7E1 assay 9
7. Targeted deep sequencing 10
8. RGEN-RFLP 10
9. Protoplast regeneration 11

RESULTS 14
1. Targeted gene modification in plant protoplasts using RGEN RNPs 14
a. Targeted gene disruption in protoplasts of plants 14
b. Analysis of off-target effects 20
c. Comparison of the delivery methods of RGEN 22
2. Targeted gene knockout and whole plant regeneration in lettuce using RGEN RNPs 26
a. Generation of BIN2 gene knockout lettuce 26
b. Analysis of off-target effects in regenerated plantlets 33
c. Germline transmission of mutant-alleles 38

DISCUSSION 44


CHAPTER 2. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells 48

INTRODUCTION 49

MATERIALS AND METHODS 53
1. Cas9 and Cpf1 ribonucleoproteins 53
2. Plasmids encoding Cpf1 and crRNAs 54
3. Cell culture and transfection conditions 54
4. Isolation of mutant clones 55

RESULTS 56
1. Genome editing in human cells using Cpf1 system 56
a. Optimization of Cpf1 system 56
b. Isolation of mutant clones 65
c. Comparison of the mutation frequencies obtained with Cpf1 and SpCas9 67
2. Analysis of target specificity of Cpf1 71
a. Analysis of target specificity of Cpf1 using mismatched crRNAs 71
b. Genome-wide target specificity of Cpf1 74
c. Analysis of target specificity of Cpf1 using truncated crRNAs 82

DISCUSSION 84

REFERENCES 88
ABSTRACT IN KOREAN 102
-
dc.formatapplication/pdf-
dc.format.extent3103209 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectGenome engineering-
dc.subjectProgrammable nucleases-
dc.subjectClustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-
dc.subjectRNA-guided Endonucleases (RGENs)-
dc.subjectCpf1-
dc.subject.ddc540-
dc.titleHighly specific genome editing in human cells and plants using CRISPR systems-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pages102-
dc.contributor.affiliation자연과학대학 화학부-
dc.date.awarded2017-02-
Appears in Collections:
College of Natural Sciences (자연과학대학)Dept. of Chemistry (화학부)Theses (Ph.D. / Sc.D._화학부)
Files in This Item:
  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse