Cas9-sgRNA 리보핵산단백질복합체를 이용한 류코노스톡 시트륨 유전체 편집기술 개발

김슬아 충북대학교 2022 국내박사

Lactic acid bacteria (LAB) have been used as starters or probiotics for foods fermentation and cell factories for bioresource production. Leuconostoc citreum is a gram positive, facultative anaerobic, and hetero-fermentative LAB frequently found in fermented vegetables, and has been used as a starter culture in kimchi fermentation. To improve their probiotic properties, removal of undesired characteristics and addition of new desirable properties by efficient genome engineering tool is necessary for probiotics or industrial use. Recently, the CRISPR-Cas9 system which is bacterial defense mechanism against virus infection has been widely used as a powerful genome editing tools in various cell types such as animal, plant, fungi, and yeast. Delivery of Cas9 by ribonucleoprotein complexes (RNP), in which recombinant Cas9 protein preassembled with sgRNA, are preferable methods due to easy production, low off-target effect, and no requirement of vector system. Although RNP system have been used for various eukaryotes cells, no studies have been applied to LAB due to limitations such as complex cell wall structure, intracellular nucleases, and lack of recombinase. Therefore, the aim of this study is development of efficient genome editing system in L. citreum using Cas9-sgRNA ribonucleoprotein complexes. To achieve the goal, RNP concentration and electroporation conditions were optimized for introducing RNP into L. citreum, and thiolated DNA and RecT recombinase originated from Lactobacillus plantarum WCFS1 were employed to increase the repair efficiency. In addition, to support above experiments, complete genome sequences of L. citreum EFEL2700 was analyzed and species/strain-specific monitoring technique using real-time qPCR was established. In chapter 2, the complete genome sequence of L. citreum EFEL2700 was analyzed by Illumina and Pacbio sequencing. As results, this strain has 1 chromosome and 4 plasmids (total 1,923,830 bp) with a G + C content of 39.0% and high homology (≥ 99%) in ANI value to the reference strain L. citreum KM 20. The KEGG analysis of metabolic pathways showed a typical hetero-type lactic acid fermentation with biosynthetic route for coenzyme A, and terpenoid backbone including isoflavonoid and riboflavin. No core genes for primary metabolism were present in plasmid 4 and it could be eliminated to create an efficient host for gene transformation. In chapter 3, species/strain-specific quantitative PCR for Leuconostoc spp. were developed and they were applied for identification and enumeration of starter culture in fermented food. As results, primers, NlacF and NlacR, for detecting L. lactis strains were developed based on pan-genome analysis. PCR analysis against genomic DNA of various LAB exhibited the primer set made a specific binding with genomic DNA of L. lactis species. The qPCR with these primers was proved to enumerate accurate population of L. lactis during kimchi fermentation. In addition, three primer sets for detecting L. mesenteroides DRC1506 were developed based on pan-genome analysis. PCR analysis against genomic DNA of various LAB exhibited the primer set made a specific binding with genomic DNA of L. mesenteroides DRC1506 strains. The qPCR method was proved to count the L. mesenteroides DRC1506 during kimchi fermentation. In chapter 4, genome editing of L. citreum via Cas9-sgRNA ribonucleoprotein complexes with RecT was conducted. For this, dextransucrase gene was selected as a target DNA and pre-assembled RNP complexes with RecT were introduced to L. citreum by electroporation. As result, dextran-free colonies were successfully obtained on sucrose agar medium, and it was found that single nucleotide deletion occurred resulting in gene knock-out by a frame shift. To increase the mutation efficiency, thiolated donor DNA and RecT recombinase were employed and the efficiency was increased up to 3.9%. In chapter 5, characteristics of L. citreum Δdsr mutant obtained in the above chapter were analyzed and its fermentation profile was investigated during dongchimi fermentation. As result, the cell growth rate and pH change of the mutant were not significantly different (p < 0·05) compared to the wild type during fermentation. When the mutant strain was inoculated as a starter in dongchimi, the mutant slowly metabolized sucrose resulting in significant reduction (6.5-folds) of dextran production compared to the wild type during fermentation. NMR analysis revealed that the polymer produced by the mutant was alternan having α-(1→3) (39.5%) or α-(1→6)-linkage (60.5%) possibly synthesized by alternansucrase. Meanwhile, viable cell counts and pH change were not significantly different (p < 0·05) compared to the wild type. In conclusion, for the first time in LAB, dextransucrase gene in L. citreum EFEL2700 was deleted by electroporation of Cas9-sgRNA ribonucleoprotein complexes and the knock-out efficiency was increased up to 3.9% by employing both thiolated donor DNA and RecT recombinase. As a universal genome engineering tool customized for LAB, this RNP complex method can be applicable to other LAB such as Bifidobacterium, Lactobacillus, and Lactococcus beyond the genus barrier. In addition, the DNA-free tools used in this study can be regarded as non-LMO technology, when no exogenous DNA were used.

Development of the Cas9-gRNA ribonucleoprotein-meditated editing for inactivation of hemolysin gene in Lactiplantibacillus plantarum

김혜준 서울대학교 대학원 2024 국내석사

Lactiplantibacillus plantarum is lactic acid bacteria commonly used as a probiotics. For many years, probiotic products using L. plantarum have been widely used for their health benefits without warning of side effects. However, many cases have reported bacteremia caused by probiotics in susceptible patients, and it can cause sepsis when the bacteria that produce the toxin (supposed hemolysin) go into the patient’s bloodstream. Thus, the aim of this study is the construction of a hemolysin gene (hemolysin Ⅲ) inactivated L. plantarum and the development of a novel editing system in L. plantarum using Cas9-gRNA ribonucleoprotein. For this, ribonucleoprotein targeting hemolysin Ⅲ transformed into L. plantarum with recombinase RecE/T and double-stranded DNA. As a result, hemolysin Ⅲ-inactivated mutants were obtained. In sequencing analysis, 50 bp deletion and stop codon insertion occurred on hemolysin Ⅲ. In conclusion, the hemolysin Ⅲ was successfully inactivated in L. plantarum by the cas9-gRNA ribonucleoprotein-meditated editing that is time-saving and low-cytotoxicity. In addition, the hemolytic activity test showed a 21.1% and 34.7% reduced activity of △hlyIII mutants compared to wild type in L. plantarum WCFS1 and SPC L. plantarum, respectively. Lactiplantibillus plantarum은 프로바이오틱스에 널리 이용되는 균주로, 건강에 많은 도움을 주기 때문에 오랜 시간 동안 부작용에 대한 경고 없이 이용되어 왔다. 하지만, 프로바이오틱스를 섭취한 면역저하자가 균세균혈증에 감염된 많은 사례들이 보고되었으며, 이때 독소 (용혈소로 추정)를 생성하는 균이 환자의 혈류 안으로 들어가면 심한 경우 패혈증을 일으킬 수 있다. 따라서, 이 연구는 용혈소 유전자 (hemolysin III)가 불활성화된 L. plantarum을 만들고, 이 과정에서 Cas9-gRNA 리보핵산단백질복합체를 이용한 새로운 편집 방식을 L. plantarum에 적용하는 것이 목표이다. 이를 위해, hemolysin III 유전자를 표적으로 하는 리보핵산단백질복합체를 재조합효소 RecE/T, 이중 가닥 DNA와 함께 L. plantarum 안으로 형질전환 해주었다. 그 결과, hemolysin III가 불활성화된 돌연변이를 얻을 수 있었으며, 서열 분석 결과를 통해 hemolysin III에서 50 bp 삭제와 종결 코돈 삽입이 일어난 것을 확인하였다. 결론적으로, 시간이 절약되고 세포 독성이 낮은Cas9-gRNA 리보핵산단백질 복합체를 이용한 편집 방식으로, L. plantarum에서 hemolysin III 유전자를 성공적으로 불활성화 시켰다. 또한, 용혈 활성 시험 결과, ∆hlyIII 돌연변이가 야생형과 비교하여 L. plantarum WCFS1 and SPC L. plantarum에서 각각 21.1%, 34.7%의 감소된 유리 헤모글로빈 농도를 보이는 것을 확인하였다.

Analysis of protein-protein interactions among heterogeneous nuclear ribonucleoproteins and their roles in IRES-dependent translation

김종헌 포항공과대학교 2003 국내박사

DNA 손상시 유도되는 hnRNP-K의 수모화에 의한 세포 주기 중지 조절에 관한 연구 : Studies on the control of p53 mediated cell-cycle arrest by hnRNP-K sumoylation in response to DNA damage

이성원 서울대학교 대학원 2013 국내박사

P53은 세포의 삶과 죽음에 관련된 다양한 신호전달 과정에 관여한다. P53은 전사인자로써 세포 손상시 다양한 표적 유전자의 발현을 조절하고 동시에 세포 주기 중지, DNA 복구 그리고 세포 사멸과 같은 다양한 반응을 유도한다. DNA 손상에 반응하여 활성화된 p53은 결과적으로 G1/S 혹은 G2/M 시기의 세포 주기 정지를 유도하는데, G1/S 시기의 정지는 cyclin 의존적 활성효소 억제인자인 p21에 의해서 주요하게 유도된다. Small Ubiquitin-related modifier (SUMO, 수모)는 유비퀴틴 유사 단백질로써 다양한 세포내 단백질에 결합한다. 수모는 유비퀴틴과 마찬가지로 E1 활성효소 (SAE1/SAE2), E2 접합효소 (Ubc9), E3 결합효소 (PIASs)에 의한 연쇄 반응 효소 작용에 의해 표적 단백질에 결합한다. 결합된 수모는 수모 특이적 단백질 분해효소에 의해 제거된다. 이런 가역적 수모화는 전사과정, 핵내 이동과정, 신호전달체계와 같은 다양한 세포내 과정을 조절할 수 있다. Heterogeneous nuclear ribonucleoprotein-K (hnRNP-K)는 RNA 결합 단백질로써 염색질 재형성, 전사과정, mRNA 스플라이싱, 번역과정과 같은 다양한 세포내 과정과 연관되어 있다. hnRNP-K는 정상적인 상태에서는 HDM2에 의해서 유비퀴틴화되어 proteosome에 의해 분해된다. 그러나 세포내에 DNA 손상이 일어나면 hnRNP-K는 안정화되고 p53의 전사 활성 인자로 작용하여 세포 주기 정지를 일으킨다. 그러나 어떻게 hnRNP-K의 안정화와 그 기능이 조절되는지는 밝혀지지 않았다. 본 연구에서는 UV에 의해 hnRNP-K의 수모화가 유도되고, 이는 유비퀴틴화를 억제하여 hnRNP-K가 안정화됨을 밝히었다. UV 처리 후 6시간이 되면 hnRNP-K의 수모화는 현저하게 증가하고 이는 18시간만에 다시 감소하는데 이런 변화는 hnRNP-K 자체의 발현 정도와 같은 패턴으로 나타난다. 그러나 hnRNP-K의 유비퀴틴화는 UV 처리 후 6시간때에 감소했다가 다시 처음 상태로 증가한다. 이는 hnRNP-K의 수모화와 유비퀴틴화가 서로 반대적으로 관련이 있다고 할 수 있으며 결과적으로 hnRNP-K의 수모화는 자체의 안정화를 유도한다고 할 수 있겠다. 또한 IR이나 doxorubicin에 의한 다른 종류의 DNA 손상이 일어나는 상태에서도 hnRNP-K의 수모화가 유도됨을 확인함으로써, hnRNP-K의 수모화에 의한 안정화는 DNA 손상에 따라 나타나는 일반적인 반응이라 할 수 있겠다. 수모화가 되지 않는 돌연변이체와 수모화가 되어 있는 hnRNP-K 단백질을 이용하여 비교한 결과, hnRNP-K의 수모화는 HDM2와의 결합을 떨어뜨리고 p53과의 결합을 증가시키는 것을 확인할 수 있었다. 즉, 수모화된 hnRNP-K는 p53과 우선적으로 결합하는 반면, 수모화 되지 않은 hnRNP-K는 HDM2와 더 결합한다. 이처럼 hnRNP-K의 수모화는 p53이나 HDM2와의 결합에 있어서 스위치 역할을 할 수 있다. 게다가 UV에 의해 유도되는 hnRNP-K의 수모화는 p53의 전사활성능력을 증가시켜 p21의 발현을 높임으로써 세포 주기 정지를 유도한다. 이는 수모화가 되지 않는 돌연변이체와는 달리 수모화가 될 수 있는 hnRNP-K는 UV에 의해 p21의 프로모터에 동원되는 결과와 일치한다. 결론적으로 hnRNP-K의 수모화는 자체의 안정화를 증가시킬 뿐만 아니라 p53의 전사활성능력 또한 증진시킬 수 있다. PIAS3는 ATR 의존적 경향으로 hnRNP-K의 수모 E3 결합 효소로써 작용한다. 그러나 수모 특이적 단백질 분해효소인 SENP2는 UV 처리후 늦은 시기에 hnRNP-K로부터 수모를 제거하고 이는 hnRNP-K의 불안정화를 가져옴으로써 세포 주기 정지로부터 빠져나오게 한다. UV 처리 후 hnRNP-K의 수모화와 안정화가 증가했다가 감소하는 것과 마찬가지로 hnRNP-K와 PIAS3간의 결합 역시 UV 처리 후 같은 시간대에서 증가했다가 감소하며 이는 SENP2와의 결합 정도와 반대 경향으로 나타난다. 결과적으로, PIAS3와 SENP2는 UV 처리 이후 hnRNP-K의 수모화와 안정화를 시간 과정별로 서로 상반되게 조절한다. 면역 세포 화학 분석에 의하면 수모화는 hnRNP-K의 핵내로의 위치 결정에 관여한다. 정상적인 hnRNP-K는 UV에 의해 오로지 핵에만 위치하지만 수모화가 되지 않는 돌연변이체의 경우 40% 정도가 UV 처리에도 불구하고 여전히 핵과 세포질에 위치한다. 게다가 PIAS3의 감소나 SENP2의 과다발현은 UV에 의한 과다발현된 hnRNP-K의 핵내 위치를 저해한다. 그러나 세포내 hnRNP-K의 핵내 위치의 경우, UV 처리나 PIAS3 감소가 어떠한 영향도 미치지 않는 것으로 보아 수모화는 hnRNP-K의 핵내 위치에 보조적인 역할을 하는 것으로 여겨진다. 요약하여 DNA의 손상에 의해 유도되는 hnRNP-K의 수모화는 p53에 의해 야기되는 세포 주기 정지 조절에 중요한 역할을 한다.

N-Acetylglucosamine Kinase Interacts with Small Nuclear Ribonucleoprotein Polypeptide N Modulating Neurodevelopment

Binod Timalsina 동국대학교 일반대학원 2023 국내박사

NAGK(N-acetylglucosamine Kinase)는 신경 세포에서 구조적 역할로 신경 발달을 촉진하는 비정규적 기능을 가진 단백질로 확인되었다. Small nuclear ribonucleotide polypeptide N(SNRPN)은 신경 발달 및 인지 능력을 조절하는데 관련된 유전자로 잘 알려져 있다. SNRPN의 메틸화 또는 돌연변이는 Prader-Willi Syndrome (PWS)과 같은 autism spectrum disorder (ASD)의 주된 원인이 된다. 이전 연구에서 in situ 근접 결찰 분석(in situ Proximity Ligation Assay; PLA)을 통해 핵질과 수상돌기에서 NAGK와 SNRPN 사이의 상호작용이 확인되었다. 본 연구에서는 RNA 관련 SmN 단백질을 암호화하는 SNRPN의 역할, 복제 또는 삭제가 신경 발달 장애와 밀접한 관련이 있음이 조사되었다. 신경 발달의 각 단계(신경 발달에서 성숙으로의 분화)에서 NAGK와 SNRPN의 상호 작용에 대한 이해를 위해, 나는 일차 해마 배양에서 신경 발달의 초기 단계부터 후기 단계까지의 상호 작용과 그에 대한 영향을 조사하였다. NAGK와 SNRPN의 정상 발현을 정량화하기 위해 P0에서 P21까지 새끼 ICR 마우스의 뇌와 더불어 DIV 1에서 DIV 21까지 in vitro일차 해마 신경세포를 배양한 뒤 웨스턴 블롯(WB) 분석을 수행하여 비교하였다. 1차 배양 뉴런은 신경 발달의 초기(DIV 1, 3, 5) 및 후기 단계(DIV 12, 16, 21)에서 연구되었습니다. In vitro DIV 3/16 및 in vivo P3/16의 경우에서 NAGK와 SNRPN의 정상 발현이 유의하게 증가하는 일치된 결과를 나타내었다. 또한 새끼 ICR 마우스 뇌의 일차 해마 신경 세포 배양에서 이들의 상호 작용을 조사하기 위해 PLA 를 수행하였고 이 또한 immunoblotting 분석의 결과와 부합하는 것이 관찰되었다. NAGK는 신경 세포에서 수상돌기 및 축삭의 신경 성장 단계에 따라 다양한 패턴으로 SNRPN과 상호작용하는 것으로 밝혀졌다. 뉴런의 세포체와 분기점에서 DIV 3의 연결 분기 단계 동안 NAGK와 SNRPN 상호작용이 명백하게 향상되었다. PLA에서 DIV 16에서 단백질들의 상호작용이 신경 발달 단계의spinogenesis 에 관여하는 것이 결찰점의 유의미한 증가로 확인되었다. 또한, HEK293T 세포 용해물로부터의 풀다운 및 동시 면역침전 분석을 통해서도 NAGK와 SNRPN 단백질의 상호작용을 확인했다. 재조합 아데노 관련 바이러스(AAV) 벡터는 유전자 전달의 물리적 개입 기술에 비해 신경퇴화에 대한 민감도가 감소하기 때문에 뉴런에서 선호되는 유전자 도입 기술입니다. 이 연구에서 나는 AAV 벡터에서 NAGK 및 SNRPN 유전자를 클로닝하고, 신경 발달의 초기 및 후기 단계 동안 1차 신경 세포 배양에서 과발현, 녹다운 및 결합 효과를 관찰했다. DIV 4 및 DIV 14에서 신경 세포의 형태를 분석하고 PLA를 수행하여 NAGK-SNRPN 상호 작용을 확인했다. NAGK와 SNRPN의 과발현은 신경 발달의 초기 단계와 후기 단계 모두에서 신경세포 분기를 크게 증가시키고 신경 복잡성을 촉고, NAGK 및 SNRPN 단백질의 녹다운은 두 단계 모두에서 신경 분기 및 파생물을 상당히 감소시켰다. 또한 NAGK 및 SNRPN 과발현과 녹다운된 신경세포에 대한 PLA를 통해 상호 작용의 증감을 분석하여 일치된 결과를 확인했다. 또한 신경돌기에서 SNRPN 및 NAGK와 함께 세포질 다이네인 성분, 특히 다이네인 경쇄 로드블록 1형(DYNLRB1)을 공동 국소화하여 세포내 소포 전송 기능을 확인했다. NAGK, SNRPN 및 DYNLRB1 간의 상호작용은 PLA에 의해 확인되었으며, 이는 신경 발달 단계 중 DIV 3 및 16에서 상당히 상향 조절되었습니다. 이들 상호작용은 멜라노좀 수송 작용을 특징으로 하는 흑색종 세포주(SK-MEL-31)에서 추가로 검증되었다. 면역세포화학(ICC) 및 PLA를 통해 미세소관에서 DYNLRB1과 함께 국소화된 NAGK 및 SNRPN 반점을 분석하기 위해 Dynein 억제제인 ciliobrevin D 를 사용하여 역행성 멜라닌소체 수송을 억제시켰다. 대조적으로, 노코다졸을 사용한 미세소관의 파괴는 NAGK, SNRPN 및 DYNLRB1의 분산된 공동 국소화를 드러냈다. 이러한 발견은 NAGK와 SNRPN이 뉴런의 소포 밀매에 관여하여 뉴런 분기 및 파생물을 향상시킨다는 것을 강력하게 뒷받침한다. 결론적으로, 나는 NAGK와 SNRPN 사이의 상호작용이 신경발달 조절에 결정적이라는 것을 발견했다. N-acetylglucosamine Kinase (NAGK) has been identified as an anchor protein that facilitates neurodevelopment with its non-canonical structural role in neurons. Small nuclear ribonucleoprotein polypeptide N (SNRPN) is an imprinted gene that regulates neurodevelopment and cognitive ability. Methylation or mutations in SNRPN can lead to the autism spectrum disorders (ASD) such as Prader-Willi syndrome (PWS). Previously, the interaction between NAGK and SNRPN in the nucleoplasm and dendrites was confirmed by in situ proximity ligation assay (PLA). In this study, I investigated the role of SNRPN, which encodes the RNA-associated SmN protein, duplications or deletions of which are strongly associated with neurodevelopmental disabilities. Since the interaction of NAGK and SNRPN in different neuronal developmental stages (neuronal differentiation to maturation) is poorly understood, I investigated their interaction and effect from the early to late stages of neurodevelopment in the primary hippocampal culture. I performed a western blot (WB) analysis to quantify the normal expression of NAGK and SNRPN in the mouse pup brain from P0 to P21, followed by the in vitro primary hippocampal culture from DIV 1 to DIV 21. The primary culture neurons were studied in the early (DIV 1, 3, 5) and late phases (DIV 12, 16, 21) of neurodevelopment. The normal expression of NAGK and SNRPN was significantly elevated at the DIV 3/16 in vitro and P3/16 in vivo. PLA was performed to investigate their interaction in primary hippocampal cultures from rat pup brains. NAGK was found to interact with SNRPN in a variable pattern depending on the stages of neuronal growth from the cell body to the dendrites/axons. NAGK and SNRPN interaction was distinctly upregulated during the neuronal branching stage at DIV 3 on the cell body and branching points of the neuron. Similar increments of the PLA dots were observed at DIV 16 on the neuronal process exhibiting its interaction during the spinogenesis stage. In addition, the interaction of NAGK and SNRPN proteins was confirmed via the pull-down and co-immunoprecipitation assays from HEK293T cell lysates. Recombinant Adeno-associated virus (AAV) vectors are the preferred gene inoculation technique in neurons due to their reduced susceptibility to neurodegeneration compared to physical intervention techniques of gene transfer. In this study, I cloned the NAGK and SNRPN genes in the AAV vectors and observed their overexpression, knockdown, and combined effects in primary neuronal culture during the early and late phases of neurodevelopment. The morphology of the neurons at DIV 4 and DIV 14 was analyzed and PLA was performed to verify the NAGK-SNRPN interaction. Overexpression of NAGK and SNRPN significantly increased the branching and promoted neuronal complexity in both the early and late stages of neurodevelopment. However, the knockdown of the NAGK and SNRPN proteins significantly reduced neuronal branching and outgrowth in both stages. PLA further validated the overexpression and knockdown effects by showing increased interactions during NAGK and SNRPN overexpression in the neuron and vice-versa. Additionally, I confirmed the intracellular vesicular trafficking of NAGK along with SNRPN in neurites during neuritogenesis and synaptogenesis stages by co-localizing cytoplasmic dynein components, specifically dynein light chain roadblock type 1 (DYNLRB1). The interaction between NAGK, SNRPN, and DYNLRB1 was confirmed by PLA, which was significantly upregulated at crucial stages of neurodevelopment (DIV 3/16). The interaction was further validated in the melanoma cell line (SK-MEL-31), considering the melanosome transport machinery. Dynein inhibitor (ciliobrevin D) was used to inhibit retrograde melanosome transport, which demonstrated the colocalized NAGK and SNRPN puncta with DYNLRB1 on microtubules through immunocytochemistry (ICC) and PLA. In contrast, disruption of the microtubule using nocodazole revealed distributed colocalization of NAGK, SNRPN, and DYNLRB1. These findings strongly support the involvement of NAGK and SNRPN in vesicular trafficking in the neurons, enhancing neuronal branching and outgrowth. In addition, the introduction of the NAGK and SNRPN in the SNRPN deletion model of PWS patient-derived induced pluripotent stem cell (iPSC) neurons, significantly promoted axodendritic development. To conclude, I found that the interaction between NAGK and SNRPN is crucial in regulating neurodevelopment.

Bioinformatic, Genetic, and Biochemical Investigations of the Bacterial OLE Ribonucleoprotein Complex

Fernando, Chrishan Malinda Yale University ProQuest Dissertations & Theses 2025 해외박사(DDOD)

Bacteria are under significant evolutionary pressures to maintain compact genomes. Therefore, they typically have high coding density compared to eukaryotes. As a result, large and highly structured noncoding RNAs (ncRNAs) are extremely rare in bacteria. However, the few classes of known large ncRNAs have unique and fundamental biological roles and are often catalytic. Furthermore, these ncRNAs reveal what may have been possible under a hypothetical ancient RNA world in which all major biological functions in existing organisms were performed by RNA, rather than DNA and protein. Further study of large ncRNAs is likely to reveal new insights into RNA catalysis and the biology of both modern and ancient organisms.OLE (ornate, large, extremophilic) RNAs are among the best studied bacterial large ncRNAs whose function(s) are yet unknown. Bacteria across the superphylum Bacillota are known to have OLE RNA. Experiments in the OLE-containing haloalkaliphile Halalkalibacterium halodurans have shown that OLE RNA exists in a ribonucleoprotein (RNP) complex with at least three proteins, called OapA, OapB, and OapC, where "Oap" stands for OLE-associated protein. Furthermore, it is known that genetic disruption of components of the OLE RNP complex does not produce noticeable phenotypes in standard growth conditions, but leads to growth defects under several environmental stress conditions including Mg2+ stress (>4 mM), cold stress (~20°C), ethanol stress (5%), and exposure to alternate carbon sources. However, the biological and biochemical functions of OLE RNA are still unclear. In Chapter 2, I describe my efforts to gain insights into OLE RNA biology using bioinformatics. I used a technique called phylogenetic profiling to identify protein-coding genes that either correlate or anticorrelate with the presence of OLE RNA across Bacillota. These efforts demonstrated that OLE RNA correlates with all three known OLE-associated proteins, suggesting that there is evolutionary pressure for OLE-containing organisms to also maintain these associated proteins. Additionally, I show that OLE RNA anticorrelates with a Mg2+ transporter called MpfA, which is also a distant homolog of OapA. These results suggest the OLE RNP complex may be functionally complementary with MpfA. Indeed, in Chapter 3, I show that genetic disruption of either MpfA or the OLE RNP complex leads to a Mg2+ stress phenotype that can be rescued by the other.In Chapter 4, I show work exploring connections between Mg2+ stress and central carbon metabolism using transcriptomics. I show that the Mg2+ excess growth defect can be rescued by Mn2+ supplementation. Furthermore, I show that Mg2+, cold, and ethanol stress all lead to differential regulation of similar categories of genes in ∆ole cells, suggesting perhaps that these major stress conditions may have similar or at least overlapping mechanisms.In Chapter 5, I demonstrate work describing the interactions between OLE RNA and BH3508, which is a restriction modification system cytosine methyltransferase and putative interacting partner of OLE RNA. I show that BH3508 binds OLE RNA in vitro with high affinity but low specificity, suggesting that BH3508 is unlikely to be a natural partner of the OLE RNP complex.Finally, in Chapter 6, I review major current gaps in our understanding of OLE RNA biology and novel strategies that can be used to answer outstanding questions.

Versatile application of the CRISPR-Cas system to various organisms

유지현 서울대학교 대학원 2018 국내박사

CRISPR-Cas is an adaptive immune system in bacteria and archaea. It specifically finds target through a complementary DNA-RNA hybridization and removes target DNA or RNA. A genome engineering has advanced explosively due to an introduction of CRISPR-Cas, moreover numerous studies have been accomplishing with CRISPR-Cas in most of scientific fields. Studies in below were a few instances of various applications using CRISPR-Cas. The first study developed a cancer diagnosis method in a liquid biopsy using CRISPR-Cas. CUT (CRISPR-mediated, Ultrasensitive detection of Target DNA)-PCR can detect the extremely small amounts of tumor DNA fragments among the much more abundant wild-type DNA fragments by specifically eliminating the wild-type sequences with CRISPR endonucleases and enriching tumor DNA. I computed that by using various orthologonal CRISPR endonucleases, the CUT-PCR method would be applicable to 80% of known cancer-linked substitution mutations. I further verified that CUT-PCR together with targeted deep sequencing enables detection of a broad range of oncogenes with high sensitivity Second study showed a generation of Crltd mutants by DNA-free ribonucleoprotein delivery in microalgae, Chlamydomonas reinhardtii. Crltd mutants which were generated using CRISPR-Cas had a pale-yellow coloration and reduced chlorophyll and PSI complex contents than wild type. As a results, it was supposed that CrLTD involved in a trafficking of LHCP to thylakoid membranes in a chloroplast. An analysis of off-target mutations in Crltd1 mutant confirmed that there were no unwanted mutations by an off-targeting. This study also optimized the method of the ribonucleoprotein delivery in C. reinhardtii, thus made much easy the generation of mutants by CRISPR-Cas. Finally, I tried a targeted DNA methylation using inactive Cas9 protein and DME protein which is a plant-specific DNA demethylase. A catalytic domain of DME fused to inactive Cas9, it was introduced to human cells and plants to induce a DNA demethylation at specific regions. In human cells, no significant changes were observed in the target region, KLF4 CpG island. In plant, FWA promoter region were targeted for generating fwa epi-mutants. Some of transgenic plants showed late flowering and vegetative axillary meristem, but DNA methylation levels of these plants were comparable to wild type. In addition, phenotypes of transgenic plants were not inherited in a next generation. It proved unsuccessful to develop an epi-genetic tool using dCas9-DME as present, it is expected that the tool could be improved by further experiments for an optimization. The epi-genome engineering has an advantage of changing phenotypes without DNA information changes. Therefore, dCas9-DME seems to have a potential as a tool for the epi-genome engineering.

Development of Genome Editing System in Leuconostoc citreum Using Cas9-sgRNA Ribonucleoprotein Complexes

Kim, Seul-Ah 충북대학교 2022 국내박사

Apolipoprotein E mRNA의 신경세포 가지돌기로 이동에 관한 연구

남연주 충북대학교 대학원 2008 국내석사

Purpose : Synaptic plasticity is functional and structural changes of synapse and considered as cellular mechanism of learning and memory. Transport of mRNA to dendrites and it's translation play a key role in synaptic plasticity (Jiang C and Schuman EM 2002). Staufen2, RNA-binding protein, is a component of RNP complexes and plays a key role in the transport of mRNA to neuronal dendrites. This study was aimed to isolate mRNAs which are associated with Staufen2-containing RNP complexes and to study on the transport on Apolipoprotein E (ApoE) mRNA to neuronal dendrites. Materials and methods : Myc-Staufen2 were overexpressed in cultured rat hippocampal neurons. The neuronal extracts were immunoprecipitated with anti-myc antibody and used in RNA extraction. The cDNA library was constructed by reverse transcription. By sequence of library, APOE mRNA was isolated. In order to examine the existence and localization of APOE mRNA in hippocampal neuron, in situ hybridization was performed using ApoE riboprobe and subsequently immunostained with anti-myc antibody to determine whether APOE mRNA colocalized with Staufen2-containing RNP complexes. Sindbis viral expression system was developed for overexpression of ApoE. The cultured hippocampal neurons were infected with a sindbis virus encoding mRFP-ApoE-3ʹ UTR, and used in situ hybridization to examin whether 3ʹUTR of ApoE have effects on the transport of APOE mRNA to dendrites. Results: A cDNA library of mRNA isolated from Staufen2-containing RNP complexes was constructed. APOE mRNA was localized in both the cell body and dendrites of the cultured hippocampal neuron. APOE mRNA colocalized with Staufen2-containing ribonucleoprotein (RNP) complexes in the cultured hippocampal neuron. The dendritic mRNA transports were increased in mRFP-ApoE-3ʹ UTR expressing hippocampal neurons. Conclusion: This study presents that APOE mRNA is moved to dendrites by Staufen2-mediated transport. The 3ʹ UTR of APOE mRNA affects on the this transport. 연구 목적 : 시냅스(synapse)의 기능적, 구조적인 변화인 시냅스 가소성(synaptic plasticity)은 학습과 기억의 세포학적 기본을 형성한다고 알려져 왔다. 신경세포 가지돌기(neuronal dendrite)로의 mRNA의 이동과 지역적 단백질 합성(local protein synthesis)은 시냅스 가소성에 중요한 역할을 한다(Jiang C and Schuman EM, 2002). RNA 결합단백질(RNA binding protein)인 Staufen2는 리보핵산단백질 복합체(ribonucleoprotein complex, RNP complex)에 포함되어 mRNA의 이동에 중요한 역할을 한다고 알려져 왔다. Staufen2를 포함하고 있는 리보핵산단백질 복합체에 결합하여 가지돌기로 이동하는 mRNA를 분리하고자 하였고, 이러한 mRNA들 중에서 Apolipoprotein E(ApoE) mRNA의 가지돌기로의 이동에 대하여 연구하고자 하였다. 재료 및 방법 : 배양된 rat의 해마 신경세포에서 myc-Staufen2를 과발현 시킨 뒤, anti-myc 항체로 myc-Staufen2를 면역침강 하고, 이 시료에서 mRNA를 분리하였다. 분리한 mRNA에서 cDNA library를 제조하고, sequencig하여 Staufen2 포함 리보핵산단백질 복합체와 결합한 mRNA의 존재를 확인하였다. 그리고 이러한 library에서 APOE mRNA를 분리할 수 있었다. 해마의 신경세포에서 APOE mRNA가 존재하는지 확인하기 위하여 in situ hybridization을 수행 하였다. 또한 Staufen2를 포함 리보핵산단백질 복합체와 APOE mRNA가 함께 존재하는지 알아보기 위하여 in situ hybridization과 면역형광염색(immunostaining)을 함께 사용하였다. ApoE를 과발현시키기 위하여 sindbis virus를 이용한 발현체계를 개발하였고, 신경세포의 가지돌기에서 APOE mRNA의 이동에 관련된 부위를 조사하기 위해 mRFP-ApoE-3′UTR을 발현하는 sindbis virus를 제조하고, in situ hybridization을 수행하였다. 연구 결과 : Staufen2를 포함하는 리보핵산단백질 복합체에 결합하는 mRNA에 대한 cDNA library를 제조하였다. Staufen2를 포함하고 있는 리보핵산단백질 복합체와 APOE mRNA가 결합하고 있는 것을 발견하였다. APOE mRNA는 해마의 신경세포에 다량으로 존재하고, 세포체뿐 만 아니라 가지돌기에서도 존재하는 것을 확인 할 수 있었고, Staufen2를 포함하고 있는 리보핵산단백질 복합체와 함께 존재하고 있는 것을 볼 수 있었다. 신경세포의 가지돌기에 존재하는 APOE mRNA의 이동이 APOE mRNA의 3′UTR을 과발현 시키면 증가하는 것을 볼 수 있었다. 결론: 본 연구를 통하여 APOE mRNA가 신경세포의 가지돌기로 이동하고, 이러한 이동은 APOE mRNA의 3ʹUTR에 의해 조절된다.

Base editing mediated precise genome editing in in vitro and in vivo model

박수민 성균관대학교 일반대학원 2024 국내석사

Chapter 1. The usage of transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR) technology has revolutionized genome editing in diverse organisms. Originating from bacterial immune systems, CRISPR/CRISPR-associated protein (Cas9) systems have been extensively utilized for targeted genome editing, relying on the guidance of single guide RNA (sgRNA) molecules to induce DNA double-strand breaks (DSBs) at specified loci. Despite their efficacy, DNA repair pathway such as homology-directed repair (HDR) and non-homologous end-joining (NHEJ) can result in unintended gene disruptions. To address these challenges, a new generation of genome editing tools, known as Base Editors (BEs), has emerged. Unlike conventional methods, BEs facilitate accurate point mutations without the need for DSBs or donor DNA templates. This study focuses on the development of a novel Cytosine Base Editor (CBE), named SsdA-BE, a single-stranded DNA deaminase toxin A (SsdA). By incorporating the 2XUGI structure into SsdA with the nickase Cas9 (nCas9), efficiency has been further enhanced. SsdA-BE demonstrates the ability to catalyse C-to-T conversions in DNA without causing DSBs. Furthermore, the research explores SsdA-BE's delivery including plasmids, ribonucleoproteins (RNPs), and virus-like particles (VLPs), revealing its potential as an effective editing tool. The investigation extends to the assessment of off-target effects, utilizing targeted deep sequencing to validate the specificity of SsdA-BE. In conclusion, this study introduces SsdA-BE as a novel and efficient CBE with promising applications in genome editing. The low off-target effects position SsdA-BE as a tool for precise and targeted genetic modification. Chapter 2. Krabbe disease, also known as globoid cell leukodystrophy, is a rare inherited metabolic disorder resulting from a functional deficiency of the lysosomal enzyme galactocerebrosidase (GALC). This deficiency leads to the accumulation of the toxic metabolite psychosine. Although effective treatments for Krabbe disease are not yet fully established, there have been recent reports of gene therapy based on adeno-associated viruses (AAVs). However, the transient maintenance of AAV genomes in the nucleus may result in sustained expression, potentially causing off-target effects. Moreover, the utilization of viruses for DNA delivery introduces the risk of viral vector integration into the genome of transduced cells, thereby increasing the potential for oncogenesis. Due to these concerns, the search for safer therapeutic approaches is imperative. In this paper, an alternative approach using adenine base editor-virus-like particles (ABE-VLPs) is introduced as a potential solution. These VLPs, using the Friend murine leukemia virus (FMLV) gag polyprotein as a foundation, provide a platform for encapsulating and delivering ribonucleoprotein (RNP) for targeted editing. ABE-VLPs demonstrated successful delivery and efficacy by confirming the induction of A-to-G conversion in both in vitro and in vivo experiments. With properties such as a reduced risk of off-target effects and short-lived expression, ABE-VLPs provide the basis for their effective clinical application as therapeutic tools for genetic disorders.