RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins

Handle URI:
http://hdl.handle.net/10754/316715
Title:
RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins
Authors:
Baazim, Hatoon
Abstract:
Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.
Advisors:
Mahfouz, Magdy
Committee Member:
Gehring, Christoph A.; Hamdan, Samir
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Program:
Bioscience
Issue Date:
May-2014
Type:
Thesis
Appears in Collections:
Bioscience Program; Theses; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.advisorMahfouz, Magdyen
dc.contributor.authorBaazim, Hatoonen
dc.date.accessioned2014-05-11T07:36:31Z-
dc.date.available2014-05-11T07:36:31Z-
dc.date.issued2014-05en
dc.identifier.urihttp://hdl.handle.net/10754/316715en
dc.description.abstractDeveloping targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.en
dc.language.isoenen
dc.subjectCRISPR/Cas 9en
dc.subjectTranscriptional regulationen
dc.subjectactivationen
dc.subjectdCas9en
dc.subjectdCas9 - chimeric proteinsen
dc.subjectrepressionen
dc.titleRNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteinsen
dc.typeThesisen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
thesis.degree.grantorKing Abdullah University of Science and Technologyen_GB
dc.contributor.committeememberGehring, Christoph A.en
dc.contributor.committeememberHamdan, Samiren
thesis.degree.disciplineBioscienceen
thesis.degree.nameMaster of Scienceen
dc.person.id121537en
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