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A novel CRISPR/RNA-aptamer-mediated base editing system with potential therapeutic value

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TitleInfo
Title
A novel CRISPR/RNA-aptamer-mediated base editing system with potential therapeutic value
Name (type = personal)
NamePart (type = family)
Collantes
NamePart (type = given)
Juan Carlos
NamePart (type = date)
1981-
DisplayForm
Juan Carlos Collantes
Role
RoleTerm (authority = RULIB)
author
Name (type = personal)
NamePart (type = family)
Jin
NamePart (type = given)
Shengkan Victor
DisplayForm
Shengkan Victor Jin
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
chair
Name (type = personal)
NamePart (type = family)
Banerjee
NamePart (type = given)
Debabrata
DisplayForm
Debabrata Banerjee
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Fondell
NamePart (type = given)
Joseph
DisplayForm
Joseph Fondell
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Padgett
NamePart (type = given)
Richard
DisplayForm
Richard Padgett
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
outside member
Name (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (type = corporate)
NamePart
School of Graduate Studies
Role
RoleTerm (authority = RULIB)
school
TypeOfResource
Text
Genre (authority = marcgt)
theses
OriginInfo
DateCreated (qualifier = exact)
2019
DateOther (qualifier = exact); (type = degree)
2019-01
CopyrightDate (encoding = w3cdtf)
2019
Place
PlaceTerm (type = code)
xx
Language
LanguageTerm (authority = ISO639-2b); (type = code)
eng
Abstract (type = abstract)
Nuclease-dependent precise genome editing such as correction of point mutations requires introduction of targeted DNA double strand breaks (DSB) and activation of homology dependent repair (HDR), limiting its application to proliferating cells. To expand GE capabilities for therapeutic use in non-dividing somatic cells it is necessary to precisely modify nucleotides avoiding DSBs. Recently, Cas9-cytidine deaminase fusions, also known as based editors (BE), were shown to precisely modify target bases at certain genomic loci. To expand the base editing toolbox, we sought to engineer a novel base editing system based on RNA-aptamer mediated recruitment. To this end, we engineered a nuclease-deficient CRISPR/Cas9 system as a recruitment platform for non-nuclease DNA/RNA editing enzymes that catalyze C·G→T·A conversions by cytidine deamination. Targeted nucleotide modification was achieved with high precision in prokaryotic and eukaryotic cells. In bacteria, we tested our system targeting the rifampicin resistance determining region of the rpoB gene. Survival in rifampicin reached over 1000-fold higher than untreated cells. To examine whether the system can correct loss of function mutations in human genome, we treated a stably integrated non-fluorescent EGFP gene containing an A·T→G·C mutation on the chromophore sequence. Fluorescence was efficiently restored in treated cells, detecting around 10% of GFP positive cells after treatment. Next generation sequencing confirmed a G·C→A·T conversion in 60% of reads at the target position, restoring the wild type sequence, with low by-stander effect. Exome-wide sequence analysis revealed no detectable off-target effects. Targeting of endogenous loci also resulted in highly efficient nucleotide conversion at the desired C positions. We also show that our system can destroy the 3’ splice acceptor site of intron 50 in human DMD gene, potentially inducing exon 51 skipping, providing evidence of a therapeutic application to treat Duchenne muscular dystrophy. Taken together, the data show that our GE system represents a safe and promising technology for editing specific nucleotides, correcting genetic mutations or other clinically relevant applications, independent of DSB and HDR, with potential therapeutic value in non-dividing cells.
Subject (authority = RUETD)
Topic
Pharmacology, Cellular and Molecular
Subject (authority = ETD-LCSH)
Topic
Gene editing -- Therapeutic use
Subject (authority = ETD-LCSH)
Topic
CRISPR-associated protein 9
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
Identifier
ETD_9363
PhysicalDescription
Form (authority = gmd)
electronic resource
InternetMediaType
application/pdf
InternetMediaType
text/xml
Extent
1 online resource (165 pages : illustrations)
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
Note (type = statement of responsibility)
by Juan Carlos Collantes
RelatedItem (type = host)
TitleInfo
Title
School of Graduate Studies Electronic Theses and Dissertations
Identifier (type = local)
rucore10001600001
Location
PhysicalLocation (authority = marcorg); (displayLabel = Rutgers, The State University of New Jersey)
NjNbRU
Identifier (type = doi)
doi:10.7282/t3-s2dw-xk36
Genre (authority = ExL-Esploro)
ETD doctoral
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Rights

RightsDeclaration (ID = rulibRdec0006)
The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
Collantes
GivenName
Juan
MiddleName
Carlos
Role
Copyright Holder
RightsEvent
Type
Permission or license
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2018-11-16 16:43:58
AssociatedEntity
Name
Juan Collantes
Role
Copyright holder
Affiliation
Rutgers University. School of Graduate Studies
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.
RightsEvent
Type
Embargo
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2019-01-31
DateTime (encoding = w3cdtf); (qualifier = exact); (point = end)
2021-01-30
Detail
Access to this PDF has been restricted at the author's request. It will be publicly available after January 30th, 2021.
Copyright
Status
Copyright protected
Availability
Status
Open
Reason
Permission or license
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Technical

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ETD
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windows xp
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1.7
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DateCreated (point = end); (encoding = w3cdtf); (qualifier = exact)
2018-11-30T10:07:47
DateCreated (point = end); (encoding = w3cdtf); (qualifier = exact)
2018-11-30T10:07:47
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