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CRISPR-Cas9-Mediated DGCR8 Deletion in a Human Stem Cell Line and Exploration of In Vivo Applications

Gezelius, Marcus (2016) MOBT01 20152
Degree Projects in Molecular Biology
Abstract
The CRISPR-Cas9 system for genetic engineering has recently emerged as promising tool for studying gene functions in a variety of systems and organisms. Here, we are particularly interested in studying the role of DGCR8 in a human neuroepithelial-like stem (hNES) cell line. Beyond its characterized role in the microRNA biogenesis pathway, recent studies have implicated the enzyme’s involvement in other essential molecular processes for normal cell development-, progression- and maintenance. With the intent to generate multiplexed deletions in DGCR8 to establish a knockout cell line and enable future study, we use lentiviral-mediated delivery of CRISPRCas9 components with a GFP reporter using a singlevector design. Subsequent genotyping... (More)
The CRISPR-Cas9 system for genetic engineering has recently emerged as promising tool for studying gene functions in a variety of systems and organisms. Here, we are particularly interested in studying the role of DGCR8 in a human neuroepithelial-like stem (hNES) cell line. Beyond its characterized role in the microRNA biogenesis pathway, recent studies have implicated the enzyme’s involvement in other essential molecular processes for normal cell development-, progression- and maintenance. With the intent to generate multiplexed deletions in DGCR8 to establish a knockout cell line and enable future study, we use lentiviral-mediated delivery of CRISPRCas9 components with a GFP reporter using a singlevector design. Subsequent genotyping indicate the presence of multiplexed deletions in DGCR8 in the hNES cells. Furthermore, we are interested in CRISPRCas9 applications for in vivo editing. To explore the system’s functionality in a living organism, we target two genes in the mouse brain for CRISPR-Cas9 induced deletions. With genotyping approaches, we investigate the technology’s potential for in vivo genome editing in a mammalian system, and suggest the occurrence of deletions in one of the targeted genes. (Less)
Popular Abstract
Sharpening Our Molecular Scissors

With modern biological technologies, we have achieved unprecedented control over genomic information stored in our genes in the form of DNA. Encoded here are the blueprints necessary to produce all the intricate components of the living cell, including proteins. Now, with recently developed technologies for altering genetic information, we can purposely block the production of a protein of interest in cells or multicellular organisms, in order to study how it functions and what role it plays.

Here, we are interested in one particular human protein, DGCR8. Previous studies have indicated a role for this protein in the production of small molecules involved in the regulation of other genes. These small... (More)
Sharpening Our Molecular Scissors

With modern biological technologies, we have achieved unprecedented control over genomic information stored in our genes in the form of DNA. Encoded here are the blueprints necessary to produce all the intricate components of the living cell, including proteins. Now, with recently developed technologies for altering genetic information, we can purposely block the production of a protein of interest in cells or multicellular organisms, in order to study how it functions and what role it plays.

Here, we are interested in one particular human protein, DGCR8. Previous studies have indicated a role for this protein in the production of small molecules involved in the regulation of other genes. These small molecules are able to essentially turn a gene off or on, and so the role of DGCR8 in living organisms is likely crucial for determining which other proteins are made at a certain time in the body and which proteins are turned off. With a recently discovered technique for creating precise mutations –disruptions in the genetic code – our aim was to damage DGCR8 in human stem cells. By disrupting the gene for DGCR8 in these particular cells, we can see how the cells react to losing this crucial protein. In cells lacking this protein, we can also study the indirect effects on other proteins, which can tell us more of how DGCR8 interacts with other important components in the cell. The results of our study suggest that we have succeeded in deleting parts of the genetic information that codes for DGCR8. Thus, we have taken important first steps towards creating a type of cell where we can study the role of this protein, and what possible implications a loss of this protein would constitute for various diseases and developmental abnormalities.

Besides our study in stem cells, we are also interested in applying this technology for altering genetic information in a living organism. For this purpose, we use a mouse as a model system for studying how and to what degree we can manipulate genetic information in the brain. Our goal was to impair two proteins involved in the suppression of glioma, the most common type of primary brain tumors in adult humans. Studies of this type of brain tumor have also revealed that the small molecules created by our previously studied protein, DGCR8, likely play a role in its development. By removing these two proteins in the mouse brain, it would be possible to create a model for cancer development, which can further be used to study the role and interactions of various proteins, such as DGCR8, that are critical to normal development. Our results indicate that we were successful in blocking the production of one of the targeted proteins, but not the other. Although this constitutes an important first step, many challenges remain for altering genetic information in living organisms.

Advisor: Johan Jakobsson, Department of Experimental Medical Science, Lund University
Master´s Degree Project 60 credits in Molecular Biology, Specialization in Microbiology,
September 2015-June 2016
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Gezelius, Marcus
supervisor
organization
course
MOBT01 20152
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8891810
date added to LUP
2016-09-15 11:25:06
date last changed
2016-09-15 11:25:06
@misc{8891810,
  abstract     = {The CRISPR-Cas9 system for genetic engineering has recently emerged as promising tool for studying gene functions in a variety of systems and organisms. Here, we are particularly interested in studying the role of DGCR8 in a human neuroepithelial-like stem (hNES) cell line. Beyond its characterized role in the microRNA biogenesis pathway, recent studies have implicated the enzyme’s involvement in other essential molecular processes for normal cell development-, progression- and maintenance. With the intent to generate multiplexed deletions in DGCR8 to establish a knockout cell line and enable future study, we use lentiviral-mediated delivery of CRISPRCas9 components with a GFP reporter using a singlevector design. Subsequent genotyping indicate the presence of multiplexed deletions in DGCR8 in the hNES cells. Furthermore, we are interested in CRISPRCas9 applications for in vivo editing. To explore the system’s functionality in a living organism, we target two genes in the mouse brain for CRISPR-Cas9 induced deletions. With genotyping approaches, we investigate the technology’s potential for in vivo genome editing in a mammalian system, and suggest the occurrence of deletions in one of the targeted genes.},
  author       = {Gezelius, Marcus},
  language     = {eng},
  note         = {Student Paper},
  title        = {CRISPR-Cas9-Mediated DGCR8 Deletion in a Human Stem Cell Line and Exploration of In Vivo Applications},
  year         = {2016},
}