Construction, selection and application of zinc finger nucleases as a tool for genome editing in rice

다국어 초록 (Multilingual Abstract)

Zinc finger nucleases (ZFNs) are artificial restriction enzymes that can be custom-made to recognize and cleave DNA in virtually any user-defined location, leading to genome editing at user-specified genomic sites. Genome
editing by ZFNs has proven to be applicable to diverse organisms including plants. But, among monocotyledonous plants only maize has been reported to be successful (Shukla et al. 2009). With a goal of widening the
applicability of ZFNs, I intended to target a rice gene OsAP2 with ZFNs. OsAP2 encodes a member of transcription factor with AP2 domain and is known to increase grain yield particularly during drought stress when expressed under a root-specific promoter (Oh et al. 2009). With an aim of mutagenizing OsAP2 with ZFNs, I constructed 61 pairs of 4F-ZFNs, by modular assembly, with cleavage potential at sites in OsAP2 protein coding
region. Of the 61 ZFNs, 4 pairs of ZFNs showing high cleavage activity were selected by yeast single-strand annealing (SSA) assay. Of these, a pair of ZFNs with highest activity were chosen for delivery into rice
embryogenic callus via Agrobacterium. The ZF modules were linked to either wild-type (F67/F96) or obligate heterodimerization variant (F67-DAS/F96-RR or F67-RR/F96-DAS) and the two ZFN monomers recognizing adjacent half-sites were linked via 2A sequence to ensure simultaneous expression of both ZFN monomers by a single promoter. The whole ZFN sequences (excluding FokI variants, F67-DAS/F96-RR or F67-RR/F96-DAS) were rice codon-optimized. The resulting ZFN sequences
were placed downstream of two different promoters: constitutive PGD1 and root-specific RCc3. A total of 6 distinct Ti-plasmid-based vectors were then delivered into rice embryogenic callus. As a result, a total of 100 T0 transgenic plants were obtained and T1 seeds were harvested from the 15 plants. Genomic PCR analysis showed that 80% of T0 transgenic plants had integrated ZFNs in their genome but one of the PCR positive plants was found to have a partial internal deletion in ZFN. GFP analysis of dried rice seeds yielded positive signals in all transgenic plants analyzed, except one
with a partial internal deletion. Immunoblot analysis also exhibited high levels of ZFN protein expression in transgenic rice tissue. To assess ZFNs’ ability of targeted mutagenesis in rice, T7 endonuclease analysis, a mismatch selective endonuclease assay, was performed. In 5 T0 plants among those analyzed, strong evidence supporting occurrence of targeted mutation in somatic tissue was observed. For further analysis, one of the
plants was selected to clone the genomic region spanning the target site in OsAP2 and subjected to sequence analysis. Of 160 clones, 4 clones were
identified to have substitution mutations at the expected ZFN target region. In the 4 clones, a total of 6 substitution mutations were found, a mutation
frequency far exceeding that expected for spontaneous errors by Taq polymerase (between 1 x 10-4 to 2 x 10-5 errors per base pair). This strongly suggests the targeting events had occurred by ZFNs. Although the
lack of indel (insertion/deletion) appears to reflect the widespread occurrence of selective cell death as mutation accumulates at off-target sites from prolonged expression of ZFN proteins, genotyping for T1 and T2
generations will be of great help not only in finding germline indels but clarifying causes of the lack of somatic indel mutations.

국문 초록 (Abstract)

징크핑거뉴클레아제 (zinc finger nuclease; ZFN)는 최근 개발된 강력한 표적돌연변이(targeted mutagenesis) 유발 도구로서 동물시스템에서는 그 유용성이 이미 확인되어 광범위하게 활용되고 있지만, 식물에서는 쌍자엽식물 모델식물인 Arabidopsis와 작물인 담배, 옥수수, 콩에서만 그 적용이 보고되어 있을 뿐, 단자엽식물 모델식물인 벼에 대해서는 아직 보고된 바가 없다. ZFN을 이용한 표적특이적 돌연변이 유발과 같은 효과적이고 안정적인 유전자 특이적 돌연변이 유도 방법이 본 연구를 통해 벼에 대해서도 적용될 수 있음을 보여준다면 이는 대부분의 식량작물이 속한 단자엽식물의 육종 및 유전자 기능 연구에 있어서 획기적인 진전이 될 것이다. 한편, 벼의 OsAP2 유전자는 AP2 domain을 갖고 있는 전사인자로서, 특히, 뿌리특이적 프로모터에 연결하여 벼에서 발현시켰을 때 생식성장기의 가뭄저항성이 증가하여 등숙률 및 알곡수가 증가하고 그 결과 수확량이 15-25% 증가하는 특성을 보인다 (Oh et al. 2009). 본 연구에서는 단자엽식물의
모델식물인 벼의 OsAP2 유전자를 표적돌연변이시킬 목적으로 네 개의 징크핑거모듈을 가진 ZFN을 모듈어셈블리법(modular assembly method)에 의해 61쌍 합성하였고, 효모 single-strand annealing (SSA) 법을 사용하여 합성된 ZFN 중에서 표적 DNA에 대한 절단 활성이 좋은 4쌍의 ZFN을 선발하였다. 이 중 가장 절단 활성이 좋은 한 쌍의 ZFN에서 징크핑거단백질 도메인을 추출하여 여기에 야생형 FokI (F67/F96) 또는 변이형 FokI (F67-DAS/F96-RR 및 F67-RR/F96-DAS) 절단도메인과 결합하고, 이들 각각에 전신발현용 프로모터와 뿌리특이적 프로모터를 각각 연결하여 형질전환 벼를 만들어 ZFN을 발현시켰다. 그 결과, 변이형 FokI 도메인 및 전신발현용 프로모터를 장착한 ZFN을 발현하는 형질전환 벼에서 OsAP2 유전자의 표적 위치에 Taq 중합효소의 오류율 (error rate)을 훨씬 상회하는 빈도의 치환돌연변이가 유발됨을 확인하였다. 이 실험에 사용된 ZFN 유전자는 벼의 코돈에 최적화시키기 위해 합성하였는데, 이 때 한 쌍의 ZFN 유전자를 한 개의 프로모터 작용으로 동시에 같은 양 발현시킬 수 있도록 두 유전자 사이에 2A sequence를 연결하였고, 이렇게 고안 합성된 ZFN 유전자 쌍을 서로 다른 세 쌍의 FokI nuclease domain (F67/F96, F67-DAS/F96-RR, F67-RR/F96-DAS)에 연결하였다 (사정상, FokI domain 중 F67/F96만 코돈 최적화함). 합성한 세 쌍의 ZFN을 각각 2종의 프로모터 즉, 전신발현용 프로모터 PGD1 또는 뿌리특이적 프로모터 RCc3가 장착된 벼 형질전환용 운반체 내로 도입시켰다. 이렇게 하여 제작 완료된 총 6종의 운반체를 Agrobacterium을 이용하여 벼 캘러스에 형질전환시켰고 형질전환세포로부터 총 100개 식물체를 재분화시켰으며, 이 중 15 개체로부터 T1종자를 채종하였다. 총 98개 형질전환 벼 식물체에 대해 genomic PCR 분석을 한 결과 형질전환 벼 중 약 80%에서 원하는 ZFN 밴드가 나타났고 다만, 이 중 한 식물체에서 ZFN 유전자 일부에 결실이 있는 것으로 관찰되었다. 일부 식물체로부터 수확한 건조 종자에 대해 GFP 형광단백질 발현을 분석한 결과, PCR 실험에서 부분 결실된 것으로
확인된 식물체 1개를 제외한 분석한 모든 형질전환 벼에서 형광단백질이 발현되었다. 형광단백질 유전자는 ZFN 유전자와 2 kb 거리를 두고 연관되어 있다. 또 immunoblot 분석 결과, 분석한 형질전환 시료 모두에서 ZFN 단백질이 강하게 발현되고 있음을 확인하였다. OsAO2유전자 부위에서 실제로 ZFN에 의한 표적 돌연변이가 유발되는지 확인하기 위해 T7 endonuclease (T7E1) assay를 수행한 결과 분석한 시료 중 5개체에서 표적돌연변이가 유발된 증거를 확보하였고, 이 중 한 개체를 선택하여 예상되는 표적부위를 포함하는 약 300 bp를 cloning한 후 염기서열을 분석한 결과 표적 부위에 다수 (모두 6개)의 치환돌연변이가 집중되어 있음을 확인하였다. 이 치환돌연변이 빈도를 Taq 중합효소의 오류율과 비교한 결과, Taq 중합효소의 내재적 오류율을 훨씬 상회하는 것으로 나타났고, 이는 표적돌연변이가 실제로 일어났음을 강력히 시사한다고 볼 수 있다. 다만, indel (삽입/결실)이 관찰되지 않은 것은 off-targeting으로 인한 세포독성으로 인해 선택적 세포 사멸이 일어난 결과로 해석되지만, 좀 더 면밀한 분석과 indel 발견을 위해 T1및 T2종자에 대한 유전분석을 할 필요가 있다고 판단된다.

목차 (Table of Contents)

• CONTENTS
• ABSTRACT ·········································································································ⅰ
• CONTENTS ········································································································ⅴ
• LIST OF TABLES ·····························································································ⅷ
• LIST OF FIGURES ··························································································ⅸ
• LIST OF ABBREVIATIONS ···········································································ⅺ
• GENERAL INTRODUCTION··································································· 1
• LITERATURE REVIEW············································································· 5
• CHAPTER I
• Construction and Selection of Active Zinc Finger Nucleases
• 1. INTRODUCTION ······················································································· 28
• 2. MATERIALS AND METHODS
• 2.1 Plasmids ······························································································· 30
• 2.2 Library of single-finger ZFNs ························································ 35
• 2.3 E. coli and yeast strains and cloning ·········································· 35
• 2.4 Construction of a library of 4F-ZFNs ········································· 36
• 2.5 Selection of active ZFNs ·································································· 37
• 2.5.1 Construction of ZFN expression vectors ··························· 37
• 2.5.2 Construction of the yeast reporter strain ························ 40
• 2.5.2.1 Cloning of the target OsAP2 ORF
• into pUC vector
• 2.5.2.2 Transformation and selection to produce
• yeast reporter strain
• 2.5.3 Yeast single-strand annealing assay ·································· 43
• 3. RESULTS
• 3.1 Identifying potential full target cleavage sites ··························· 48
• 3.2 Construction a library of 4-finger array ····································· 49
• 3.3 Selection of active ZFNs ································································· 49
• 4. DISCUSSION ································································································ 65
• 5. REFERENCES ······························································································ 70
• CHAPTER II
• Application of Zinc Finger Nuclease Technology To Rice
• (Oryza sativa L.)
• 1. INTRODUCTION ························································································· 76
• 2. MATERIALS AND METHODS
• 2.1 Plant material and growth conditions ········································· 78
• 2.2. DNA constructs ················································································· 78
• 2.3 Transgenic plants ·············································································· 80
• 2.4 Analysis of gene targeting events ················································· 80
• 2.4.1 Genomic PCR for ZFN integration ···································· 80
• 2.4.2 Detection of GFP fluorescence ············································ 81
• 2.4.3 Immunoblot analysis ······························································ 81
• 2.4.4 T7 endonuclease assay ·························································· 81
• 3. RESULTS
• 3.1. Regeneration of transgenic plants ················································ 84
• 3.2. ZFN Integration and expression ·················································· 85
• 3.2.1. Genomic PCR ·········································································· 85
• 3.2.2. GFP assay ··············································································· 85
• 3.2.3. Immunoblot analysis ···························································· 86
• 3.3. Analysis of genome editing events ····················································· 86
• 4. DISCUSSION ································································································ 97
• 5. REFERENCES ···························································································· 102
• APPENDIX······································································································· 109
• ABSTRACT IN KOREAN············································································· 122