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.
The CRISPR/Cas Tool Kit for Genome Editing
The CRISPR/Cas Tool Kit for Genome 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 The CRISPR/Cas Tool Kit for Genome Editing write by Aftab Ahmad. This book was released on 2022-01-01. The CRISPR/Cas Tool Kit for Genome Editing available in PDF, EPUB and Kindle. This book discusses CRISPR/Cas- one of the most powerful tools available to scientists for genome editing. CRISPR/Cas is not only a genome editing tool, but researchers have also engineered it for gene regulation, genome imaging, base editing and epigenome regulations. This book describes the entire toolkit for CRISPR/Cas. The opening section gives an introduction to the technique and compares it with other genome editing tools. Further section gives a historical perspective of the tool, along with its detailed classification. The next chapters describe bioinformatic tools in CRISPR/Cas, and delivery methods for CRISPR/Cas. The book also discusses about the applications of CRISPR/Cas beyond genome editing and use of CRISPR for rewriting genetic codes. The book dedicates a section to the use of CRISPR in plants. The book culminates with a chapter on the current status, challenges and shortcomings of the CRISPR/Cas genome editing tool. The book would be highly interesting to students and researchers in molecular biology, biochemistry, biotechnology, food science, agriculture and plant sciences.
CRISPR-Cas Systems
CRISPR-Cas Systems - 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 CRISPR-Cas Systems write by Rodolphe Barrangou. This book was released on 2012-12-13. CRISPR-Cas Systems available in PDF, EPUB and Kindle. CRISPR/Cas is a recently described defense system that protects bacteria and archaea against invasion by mobile genetic elements such as viruses and plasmids. A wide spectrum of distinct CRISPR/Cas systems has been identified in at least half of the available prokaryotic genomes. On-going structural and functional analyses have resulted in a far greater insight into the functions and possible applications of these systems, although many secrets remain to be discovered. In this book, experts summarize the state of the art in this exciting field.
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.
Human Genome Editing
Human Genome 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 Human Genome Editing write by National Academies of Sciences, Engineering, and Medicine. This book was released on 2017-08-13. Human Genome Editing available in PDF, EPUB and Kindle. Genome editing is a powerful new tool for making precise alterations to an organism's genetic material. Recent scientific advances have made genome editing more efficient, precise, and flexible than ever before. These advances have spurred an explosion of interest from around the globe in the possible ways in which genome editing can improve human health. The speed at which these technologies are being developed and applied has led many policymakers and stakeholders to express concern about whether appropriate systems are in place to govern these technologies and how and when the public should be engaged in these decisions. Human Genome Editing considers important questions about the human application of genome editing including: balancing potential benefits with unintended risks, governing the use of genome editing, incorporating societal values into clinical applications and policy decisions, and respecting the inevitable differences across nations and cultures that will shape how and whether to use these new technologies. This report proposes criteria for heritable germline editing, provides conclusions on the crucial need for public education and engagement, and presents 7 general principles for the governance of human genome editing.
