Engineering Antisense Oligonucleotides for Site-directed RNA Editing with Endogenous ADAR - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook Engineering Antisense Oligonucleotides for Site-directed RNA Editing with Endogenous ADAR write by Tobias Merkle. This book was released on 2020. Engineering Antisense Oligonucleotides for Site-directed RNA Editing with Endogenous ADAR available in PDF, EPUB and Kindle.
RNA Editing Via Recruitment of Endogenous ADARs Using Circular Antisense Guide RNAs
RNA Editing Via Recruitment of Endogenous ADARs Using Circular Antisense Guide RNAs - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook RNA Editing Via Recruitment of Endogenous ADARs Using Circular Antisense Guide RNAs write by James Jen Yen. This book was released on 2021. RNA Editing Via Recruitment of Endogenous ADARs Using Circular Antisense Guide RNAs available in PDF, EPUB and Kindle. Genetic disorders collectively chronically affect 1 in 17 individuals in the world today. Currently, many of the treatments that exist for such disorders are palliative and only treat the symptoms, not the underlying cause. The small amount of approved curative treatments that do exist utilize permanent genome editing tools that carry inherent risks of permanent off-target modifications. The aim of this thesis is to develop a platform for the safe and effective repair of genetic disorders using A-I RNA editing. It has been recently shown that delivery of long antisense guide RNAs can recruit endogenous adenosine deaminase acting on RNA (ADAR) enzymes to induce RNA editing in vitro. Importantly however, this approach is unable to induce RNA editing in vivo; we hypothesized this to be a result of the short half-life of linear guide RNAs resulting from vulnerability to exonuclease attack. By engineering and delivering highly stable circular guide RNAs via AAV8, we were able to induce robust RNA editing in mice livers: we observed 53% editing in the 3'UTR of the mPCSK9 transcript in C57BL/6J mice and 12% correction of a nonsense mutation in the IDUA-W392X mouse model for type mucopolysaccharidosis type I-Hurler (MPS I-H) syndrome. Furthermore, we were able to reduce the bystander editing profile of target transcripts by engineering loop secondary structures strategically placed throughout our circular antisense guide RNAs. Altogether, our platform paves the way for safe transcript-specific RNA editing for use in gene therapy.
Structural Engineering of Adenosine Deaminases Acting on RNA with Chemically Modified Guide RNAs for Site-directed RNA Editing
Structural Engineering of Adenosine Deaminases Acting on RNA with Chemically Modified Guide RNAs for Site-directed RNA Editing - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook Structural Engineering of Adenosine Deaminases Acting on RNA with Chemically Modified Guide RNAs for Site-directed RNA Editing write by Leanna Rose Monteleone. This book was released on 2020. Structural Engineering of Adenosine Deaminases Acting on RNA with Chemically Modified Guide RNAs for Site-directed RNA Editing available in PDF, EPUB and Kindle. RNA editing is defined as the insertion, deletion, or modification of a nucleotide that changes the information content of a sequence. Adenosine deaminases acting on RNA (ADARs) can deaminate an adenosine (A) in duplex RNA to inosine (I). Cellular machinery interprets inosine as guanosine, which can result in various consequences on RNA function. A-to-I editing can alter microRNA sequences, redirect splicing, and change secondary structure. More dramatically A-to-I editing can result in a recoding event, thereby changing the amino acid at a specific position. In recent years, there has been rapidly growing interest in engineering ADARs or directing endogenous ADARs to specific G-to-A mutations linked to various diseases. The contents of this dissertation details the progress we have made, with the help of various collaborations, to use ADARs for site- directed RNA editing. In chapter 1, I review various types of RNA editing with a great focus on adenosine deamination. I emphasize ADARs biological function, substrate specificity, and the roles ADARs have in various diseases. I further discuss the structural data that is known for ADAR2 and how this knowledge has led to a better understanding of using ADARs for site-directed RNA editing. In chapter 2, I discuss the previous approaches used for site-ivdirected RNA editing with ADAR and the challenge of overcoming off-target reactions. To overcome off-target reactions, I have designed an orthogonal editing system utilizing a bump-hole strategy to prevent off-target edits. I have shown that combining bulky ADAR mutants with a chemically modified guide RNA (gRNA) achieves site-selective editing with reduced off-target edits both in vitro and in cellular assays. In chapter 3, I focus on our collaboration with Prof. Gail Mandel's laboratory at Oregon Health and Science University to study a disease-causing mutation linked to Rett Syndrome. In this approach, we have focused on rationally designing chemically modified gRNAs that could potentially recruit endogenous wild type ADARs. Our rational design utilizes the crystallography of ADAR2 constructs bound to double stranded RNA (dsRNA) that were solved by our collaborators in Prof. Andrew Fisher's laboratory. In chapter 4, I deviate from using ADARs for site-directed RNA editing to elucidate the biological role of ADAR3. ADAR3 is catalytically inactive and is exclusively located in the brain. To further understand the role of ADAR3, five mutations were incorporated to engineer an active ADAR3 (ADAR3 M3). From here, we propose that ADAR3 not only acts as a negative regulator of ADAR1 and ADAR2, but also as a direct regulator in stabilizing specific transcripts. With an active ADAR3, future studies can be done to use ADAR3 M3 or another version of an active ADAR3 for site-directed RNA editing.
Engineering a Programmable RNA Editing Toolset for Correction of Point Mutations in Vivo
Engineering a Programmable RNA Editing Toolset for Correction of Point Mutations in Vivo - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook Engineering a Programmable RNA Editing Toolset for Correction of Point Mutations in Vivo write by Dhruva Katrekar. This book was released on 2021. Engineering a Programmable RNA Editing Toolset for Correction of Point Mutations in Vivo available in PDF, EPUB and Kindle. While human genetic diseases can be caused by point mutations, insertions/deletions, chromosomal translocations or copy number variations, point mutations account for 58% of the pathogenic genetic variants causing disease. Programmable nucleases such as CRISPR-Cas are powerful tools but their use for the correction of point mutations in vivo poses some major challenges, namely, their reliance on the inefficient process of homologous recombination, threat of introducing permanent off-target mutations in the genome and immunogenicity due to their prokaryotic origin. In this dissertation, we develop and characterize an RNA editing toolset of human origin for correction of guanosine-to-adenosine mutations and premature stop codons. We engineer guide RNA to recruit exogenously expressed human adenosine deaminase acting on RNA (ADAR) enzymes to target transcripts and catalyze adenosine-to-inosine (guanosine) modifications. In a proof-of-concept study, we repair disease-causing premature stop codons and splice-site mutations in mouse models of Duchenne muscular dystrophy (DMD) and ornithine transcarbamylase (OTC) deficiency respectively, via exogenously delivered ADARs and associated guide RNA. However, exogenous delivery of ADARs leads to transcriptome-wide off-targeting, and additionally, enzymatic activity on certain RNA motifs, such as adenosines flanked by a 5' guanosine is very low, thus limiting their utility as a transcriptome engineering toolset. To solve these issues, we develop a split-ADAR system with highly improved specificity profiles and also carry out a high throughput mutagenesis screen, identifying ADAR variants with enhanced activity at adenosines flanked by a 5' guanosine. From a gene therapy perspective, recruitment of endogenous ADAR enzymes for editing a desired transcript creates minimal perturbation for the target cells as compared to exogenously delivered ADARs. Thus, we go on to engineer novel circular guide RNAs to recruit endogenous ADAR enzymes. We demonstrate its therapeutic potential by correcting a premature stop codon in a mouse model of Hurler syndrome via delivery of only circular guide RNA. Since immunogenicity against the delivery vehicle also limits efficacy of gene therapies, we develop a programmable adeno-associated virus (AAV) for gene delivery while also modifying it to evade neutralization by pre-existing antibodies in the serum.
RNA Editing
RNA Editing - read free eBook in online reader or directly download on the web page. Select files or add your book in reader. Download and read online ebook RNA Editing write by Ernesto Picardi. This book was released on 2021-08-13. RNA Editing available in PDF, EPUB and Kindle. This volume provides an overview about main RNA editing mechanisms, focusing on their functions in physiological as well as pathological conditions. Chapters guide readers through state- of-the art methodologies to investigate RNA editing through wet and dry approaches. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, RNA Editing: Methods and Protocols aims to ensure successful results in the further study of this vital field.
