Therapeutic control of off-target effect in RNA-guided gene silencing and editing = RNA 기반 유전자 조절에서 오프-타겟의 치료적 활용 방법

다국어 초록 (Multilingual Abstract)

DNA and RNA are two types of essential molecules that are vital for all living things on earth. In accordance with the central dogma, DNA acts as a template for the synthesis of RNA (transcription), and RNA directly used as a template for protein synthesis (translation). Over the past decades, researchers have been trying to control DNA, RNA, and proteins for therapeutic purposes. Most drugs, such as antibodies and aptamers, are aimed at controlling the proteins related to diseases. In the field of genetic research, regulating gene content and expression has been critical in understanding the function of genes in biological pathways and their relationship with disease phenotypes. Recent advances in technologies for controlling gene expression have improved biomedical research and expanded gene therapy possibilities. RNAi was initially discovered and widely used as a versatile tool for gene regulation via controlling mRNA stability and translation. Especially, Small interfering RNA (siRNA), a major component of RNAi, has also been widely used to control specific genes because of its ease of use based on RNA:RNA Watson-Crick base-pairings. More recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) (referred to as CRISPR/Cas9), which originates from the bacterial adaptive immune system, have emerged as a genome editing tools for targeting DNA guided by short CRISPR RNA (crRNA). Target specificity for both siRNA and CRISPR/Cas9 relies on 18-20nt RNA guided base-pairing, which have enormous possibilities for unintended target (off-target) recognitions occur via partial base-pairing. Thus, it is important to control the off-target effect in both technologies for use in therapeutic applications. Here, we develop potent therapeutic siRNAs for utilizing miRNA-like-off-target activity rather than prevent in RNAi. In contrast, we also developed a precise CRISPR/Cas9 genome editing tool by modifying crRNA since its off-target may be permanently inherited.

국문 초록 (Abstract)

DNA와 RNA 두 가지 모두 지구상의 모든 생명체에 필수적인 분자화합물이다. RNA는 서열번역을 통해 단백질 합성의 주형 가닥으로 사용되며, DNA는 RNA 전사에 사용된다. 지난 수십 년 동안, 이러한 특성을 바탕으로 인류는 DNA, RNA, 그리고 단백질 생성과 작용의 조절을 통해 이들을 치료적 목적으로 사용할 수 있도록 노력해 왔으며, 그 일환으로 항체나 앱타머 같은 대다수의 약물은 질병과 관련된 단백질을 조절하여 치료적 효과를 내는 것을 목표로 하고 있다. 이처럼 유전자 연구에서 특정 유전자 표현과 발현을 조절하는 것은 생물학적 관점에서 유전자와 질병 표현형의 관계를 이해하는 데 있어 매우 중요한데, 최근 유전자 발현 조절 기술의 향상으로 생물의학적 연구가 발전되어 유전자 치료 가능성이 확대되었다.
초기에 발견된 RNAi는 mRNA의 안정성을 제어하거나 단백질로 번역되는 것을 제어함으로써 유전자 조절을 가능하게 만들었다. 특히 RNAi의 주요 구성물질인 siRNA는 RNA:RNA 왓슨-크릭 염기쌍을 기반으로 타겟을 인식하므로, 사용의 편의성 덕분에 유전자를 조절하는 주요 물질로 사용되어 왔다. 최근에는 박테리아 후천면역 체계로부터 발견된 CRISPR/Cas9이 유전자 편집 도구로 널리 사용되고 있으며, 짧은 crRNA에 의해 표적을 인식한다는 점에서 siRNA와 공통점을 가진다. 이들 siRNA와 CRISPR/Cas9은 18-20nt RNA에 의한 염기결합으로 타겟 특이성을 가질 수 있는데, 이렇게 짧은 염기결합에 의한 타겟 인식때문에 의도하지 않은 타겟 (오프-타겟) 을 인식할 가능성이 크다. 따라서 위 두 기술 모두 치료제로써 사용하기 위해서는 오프-타겟을 제어하는 것이 매우 중요하다.
본 연구는, RNAi에서 발생할 수 있는 오프-타겟 효과인 miRNA-like activity를 억제하기보다 오히려 활용해 강력한 치료제로써 가능성을 가지는 siRNA를 구성하는 방법을 개발하였다. 이와 반대로 CRISPR/Cas9을 이용한 유전자 편집에서 발생할 수 있는 오프-타겟은, 한번 발생하게 되면 영구적으로 유전자 구성에 변경이 일어날 수 있으므로, 이 도구를 매우 정확하게 사용할 수 있도록 타겟 인식에 직접적으로 연관된 crRNA를 개선하는데 목적을 두었다.

목차 (Table of Contents)

• Table of contents
• List of Tables I
• List of Figures II
• Abbreviations IV
• Abstract - 1 -
• CHAPTER I. Overview - 3 -
• 1.1 Basic of RNAi - 4 -
• 1.1.1 miRNA and siRNA - 4 -
• 1.1.2 Potential of RNAi therapy for cancer - 9 -
• 1.2 miRNA-like off-target effects in RNAi - 11 -
• 1.2.1 Widespread miRNA-like off-target effect in RNAi method - 11 -
• 1.2.2 Widespread analysis of RNAi through miRNA-target interaction - 12 -
• 1.2.3 Strategies to reduce miRNA-like off-target repression - 14 -
• 1.3 CRISPR/Cas9 biology - 19 -
• 1.3.1 Targeted DNA DSBs using the CRISPR/Cas9 system - 22 -
• 1.3.2 Endogenous DNA repair mechanisms for genome editing - 23 -
• 1.4 Off-target effects at CRISPR/Cas9 genome editing - 28 -
• 1.4.1 Genome-wide Unbiased off-target Detection - 28 -
• 1.4.2 Cas9 protein engineering to decrease off-target activity - 31 -
• 1.4.3 guide RNA modification to reduce the off-target effects - 36 -
• 1.5 Aims of this study - 40 -
• CHAPTER II. AGO-accessible anticancer siRNAs with synergistic miRNA-like activity - 43 -
• Abstract - 45 -
• Introduction - 46 -
• Results - 48 -
• AGO CLIP analysis of HPV18 for cervical cancer - 48 -
• Tumor-suppressive activity of HPV18 E6/E7-targeted miRNAs - 49 -
• AGO CLIP-based design of antitumor mi/siRNAs against E6/E7 - 49 -
• Transcriptome-wide analyses of mi/siRNAs targeting E6/E7 - 50 -
• Suppression of cervical cancer cells by 206/E7 and 218/E7 - 53 -
• AGO CLIP-based strategy of designing mi/siRNAs for cancer treatment - 55 -
• Validation of mi/siRNAs designed for ovarian and breast cancers - 55 -
• Materials and Methods - 59 -
• Bioinformatics and statistical analysis - 59 -
• AGO CLIP analysis on HPV18 - 59 -
• miRNA profile analysis in cervical cancer - 59 -
• Small RNA synthesis - 59 -
• Cell culture and transfection - 60 -
• Cell death analysis - 60 -
• Survival analysis - 60 -
• Wound-healing assay - 60 -
• Invasion assay - 60 -
• Construction of luciferase reporters - 60 -
• Luciferase reporter assay - 61 -
• Immunoblotting - 61 -
• Quantitative RT-PCR analysis - 61 -
• In vitro Ago2 cleavage assay - 61 -
• RNA-seq analysis - 61 -
• Cumulative distribution function and volcano plot analyses - 61 -
• GO analysis - 62 -
• DNA fragmentation assay with flow cytometry - 62 -
• Xenograft mouse model and siRNA treatment - 62 -
• Construction of mi/siRNA web server for cervica, ovarian, and breast cancer - 62 -
• References - 63 -
• CHAPTER III. AGO CLIP based imputation of potent siRNA sequences targeting SARS-CoV-2 with antifibrotic miRNA-like activity - 79 -
• Abstract - 80 -
• Introduction - 81 -
• Material and Methods - 82 -
• Bioinformatics and statistical analyses - 82 -
• Sequence analysis of SARS-CoV-2 - 82 -
• Meta-analysis of AGO CLIP data for SARS-CoV-2 - 82 -
• Calculation of the exposure probability in SARS-CoV-2 - 83 -
• RNA synthesis - 83 -
• Cell culture, transfection, and treatment - 83 -
• Construction of luciferase reporter constructs - 83 -
• Luciferase reporter assays - 84 -
• RNA-seq analysis - 84 -
• CDF and volcano plot analyses - 84 -
• Gene ontology analysis - 84 -
• Overlap analysis of 27/RdRP and miR-27a downregulated targets - 84 -
• Prediction of the SARS-CoV-2 nsp12 secondary structure - 85 -
• Quantitative RT-PCR analysis - 85 -
• Quantification of SARS-CoV-2 target RNAs silenced by siRNA - 85 -
• Construction of the siRNA webserver for SARS-CoV-2 - 86 -
• Results and discussion - 86 -
• AGO CLIP-based imputation of AGO accessibility in SARS-CoV-2 - 86 -
• Designing potent SARS-CoV-2 siRNAs with antifibrotic miRNA-like activity - 86 -
• Validation of siRNAs targeting SARS-CoV-2 with antifibrotic miRNA activity - 86 -
• Transcriptome-wide validation of 27/RdRP for antifibrotic miRNA-like activity - 89 -
• Validation of the antifibrotic siRNA activity of 27/RdRP targeting SARS-CoV-2 - 93 -
• Conclusion - 94 -
• References - 94 -
• Chapter IV. Combined modification of abasic substitution and extension in guide RNAs improves target specificity of CRISPR-Cas9 - 104 -
• Abstract - 105 -
• Introduction - 106 -
• Materials and methods - 111 -
• Genomic DNA extraction and target amplification - 111 -
• In vitro Cas9 cleavage assay - 111 -
• Cell culture and Transfection - 112 -
• Construction of surrogate reporters to measure genome editing efficiency - 113 -
• Cell-based genome editing efficiency measured by flow cytometry - 113 -
• Oligonucleotide synthesis for Target-biased random libraries - 114 -
• DESTINY-seq - 115 -
• Analysis of DESTINY-seq data - 117 -
• Results - 128 -
• Abasic substitution limits target complementarity maintaining Cas9 binding sites - 128 -
• w17 crRNA prevents off-target effect as tru-crRNA - 138 -
• Abasic extension increases on-target specificity without extra base-pairing effect - 143 -
• wX20 reduces off-target at the level of GGX20 - 148 -
• wXw crRNAs broadly increase specificity of Cas9 - 152 -
• Discovery of positional preference at CRISPR/Cas9 using target biased random library by sequencing (DESTINY-seq) - 159 -
• Discussion - 176 -
• References - 179 -
• CONCLUSION - 199 -
• ABSTRACT IN KOREAN - 202 -
• ACKNOWLEDGEMENTS - 204 -