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Adaptation of CRISPR/Cas9 to improve the experimental utility of the model system schizosaccharomyces pombe

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TitleInfo
Title
Adaptation of CRISPR/Cas9 to improve the experimental utility of the model system schizosaccharomyces pombe
Name (type = personal)
NamePart (type = family)
Ciccaglione
NamePart (type = given)
Keith M.
NamePart (type = date)
1988-
DisplayForm
Keith M. Ciccaglione
Role
RoleTerm (authority = RULIB)
author
Name (type = personal)
NamePart (type = family)
Zaratiegui
NamePart (type = given)
Mikel
DisplayForm
Mikel Zaratiegui
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
chair
Name (type = personal)
NamePart (type = family)
Brill
NamePart (type = given)
Steven
DisplayForm
Steven Brill
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Copeland
NamePart (type = given)
Paul
DisplayForm
Paul Copeland
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (type = corporate)
NamePart
Graduate School - New Brunswick
Role
RoleTerm (authority = RULIB)
school
TypeOfResource
Text
Genre (authority = marcgt)
theses
OriginInfo
DateCreated (encoding = w3cdtf); (qualifier = exact)
2015
DateOther (qualifier = exact); (type = degree)
2015-10
CopyrightDate (encoding = w3cdtf); (qualifier = exact)
2015
Place
PlaceTerm (type = code)
xx
Language
LanguageTerm (authority = ISO639-2b); (type = code)
eng
Abstract (type = abstract)
Schizosaccharomyces pombe is a model organism that is utilized in several areas of research including DNA and chromatin biology. However, methods for genomic manipulation of the organism are outdated and cumbersome. The CRISPR/Cas9 toolset was recently developed from the prokaryotic type II CRISPR-associated system (a form of adaptive immunity) and has been shown to be successful at site-specific mutagenesis and modulation of gene expression in multiple eukaryotic organisms. We sought to modify the two components of this instrument, Cas9 and short-guide RNA (sgRNA), for effective use in S. pombe. As proof of concept, the ade6 gene was targeted for CRISPR/Cas9 mutagenesis by employing two constructs: one with an adh1 expressed Cas9 and one with sgRNA expressed by rrk1, the promoter for K RNA, and precisely terminated by a Hammerhead ribozyme. The ade6 gene, a long terminal repeat (LTR) sequence of the retrotransposons Tf1 and Tf2, and TER1 were also targeted by the rrk1 sgRNA and the catalytically null Cas9 (dCas) for site-specific enrichment of DNA sequence through chromatin immunoprecipitation. We found some Cas9 expressing vectors were inactivated by random mutation and studied this phenomenon using the inducible nmt1 promoter (no mRNA transcription in thiamine). Cultures with either nmt1:Cas9 or nmt1:dCas9 repressed by thiamine grew 4.4 to 7 times (p-value= 2.1x10-15 and 2.2x10-16, respectively) faster than those with full-expression. Toxicity was not attributable to the catalytic activity of Cas9 and further investigation is required. Cas9 and sgRNA were combined into a single expression vector to reduce exposure to inactivating mutations and yielded mutagenesis efficiencies of 86-92%. Also, by reducing expression of nmt1:dCas9 in thiamine, we achieved 50% to 300% enrichment of genomic target sites. The described rrk1 expressed sgRNA, with Cas9 and dCas9, will enable more efficient genomic manipulation of S. pombe.
Subject (authority = RUETD)
Topic
Microbiology and Molecular Genetics
Subject (authority = ETD-LCSH)
Topic
Gene expression
Subject (authority = ETD-LCSH)
Topic
Schizosaccharomyces pombe
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
Identifier
ETD_6782
PhysicalDescription
Form (authority = gmd)
electronic resource
InternetMediaType
application/pdf
InternetMediaType
text/xml
Extent
1 online resource (viii, 52 p. : ill.)
Note (type = degree)
M.S.
Note (type = bibliography)
Includes bibliographical references
Note (type = statement of responsibility)
by Keith M. Ciccaglione
RelatedItem (type = host)
TitleInfo
Title
Graduate School - New Brunswick Electronic Theses and Dissertations
Identifier (type = local)
rucore19991600001
Location
PhysicalLocation (authority = marcorg); (displayLabel = Rutgers, The State University of New Jersey)
NjNbRU
Identifier (type = doi)
doi:10.7282/T3N58P92
Genre (authority = ExL-Esploro)
ETD graduate
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Rights

RightsDeclaration (ID = rulibRdec0006)
The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
Ciccaglione
GivenName
Keith
MiddleName
M.
Role
Copyright Holder
RightsEvent
Type
Permission or license
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2015-09-25 07:52:06
AssociatedEntity
Name
Keith Ciccaglione
Role
Copyright holder
Affiliation
Rutgers University. Graduate School - New Brunswick
AssociatedObject
Type
License
Name
Author Agreement License
Detail
I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
Copyright
Status
Copyright protected
Availability
Status
Open
Reason
Permission or license
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Technical

RULTechMD (ID = TECHNICAL1)
ContentModel
ETD
OperatingSystem (VERSION = 5.1)
windows xp
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