Matching Items (2)
Filtering by
- Creators: School of Life Sciences
Description
Extensive efforts have been made to develop efficient and low-cost methods for diagnostics to identify molecular biomarkers that are linked to a wide array of conditions, including cancer. A highly developed method includes utilizing the gene-editing enzyme CRISPR-Cas12a (Cpf1), which demonstrates double-stranded DNase activity with RuvC catalytic domain with high sensitivity and specificity. This DNase activity is RNA-guided and requires a T-rich PAM site on the target sequence for functional cleavage. There have been recent efforts to utilize this DNase activity of Cas12a by combining it with isothermal amplification and analysis by lateral strip tests. This project examined CRISPR-based early detection of microRNA biomarkers. MicroRNA are short RNA molecules that have large roles in post-transcriptional gene regulation. However, due the short length of microRNA and its single-stranded nature, it is challenging to use Cas12a for microRNA detection using existing methods. Thus, this project investigated the potential of two microRNA detection strategies for recognition by CRISPR-Cas12a. These methods were microRNA-splinted ligation with polymerase chain reaction (PCR) and MicroRNA-specific reverse transcriptase PCR (RT-PCR). Gel imaging demonstrated effective amplification of ligated DNA through microRNA-splinted ligation with PCR/RPA. In addition, lateral strips tests showed effective cleavage of the target sequences by Cas12a. However, RT-PCR method demonstrated low amplification by PCR and inefficient poly(A) elongation. This project paves the way for the detection of an extensive range of microRNA biomarkers that are linked to an array of diseases. Future directions include analysis and modifications of RT-PCR method to improve experimental results, extending these detection methods to a larger range of microRNA sequences, and eventually utilizing them for detection in human samples.
ContributorsStaren, Michael Steven (Author) / Green, Alexander (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Diehnelt, Chris (Committee member) / School of Life Sciences (Contributor) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Advancements in science and technology, particularly in the field of genome editing, hold significant potential to change how future generations will treat disease and may fundamentally change what it means to be human. There are concerns by scientists and non-scientists about how to explore the values and perceptions of the public regarding the implications of new technologies. Use of participatory Technology Assessment (pTA) has arisen as a type of interactive group discussion to disseminate information about technology and collect non-scientists' perceptions of the value, impact or usefulness of a technology and potential ethical issues or consequences to be considered. There is no one size fits all model of pTA; several are discussed in this paper, but there are similarities between them such as the structure of engagement or recruitment criteria. It is important to note a difference in public understanding of science and public engagement with science as it relates to the structure and execution of pTA. This study was undertaken to evaluate pTA as a tool to explore perceptions, values and opinions regarding a case study of CRISPR-Cas9, a tool for genome editing, among ASU Barrett undergraduate students.
ContributorsChapin, Natalie Ann (Author) / Brian, Jennifer (Thesis director) / Bennett, Ira (Committee member) / School of Life Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12