title: CRISPR-based self-cleaving mechanism for controllable gene delivery in human cells.
abstract: Controllable gene delivery via vector-based systems remains a formidable challenge in mammalian synthetic biology and a desirable asset in gene therapy applications. Here, we introduce a methodology to control the copies and residence time of a gene product delivered in host human cells but also selectively disrupt fragments of the delivery vehicle. A crucial element of the proposed system is the CRISPR protein Cas9. Upon delivery, Cas9 guided by a custom RNA sequence cleaves the delivery vector at strategically placed targets thereby inactivating a co-expressed gene of interest. Importantly, using experiments in human embryonic kidney cells, we show that specific parameters of the system can be adjusted to fine-tune the delivery properties. We envision future applications in complex synthetic biology architectures, gene therapy and trace-free delivery.
authors.affiliation: Bioengineering Department, University of Texas at Dallas, Richardson, TX 75080, USA Electrical Engineering Department, University of Texas at Dallas, Richardson, TX 75080, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Moore
authors.affiliation: Bioengineering Department, University of Texas at Dallas, Richardson, TX 75080, USA.
authors.forename: Alec
authors.initials: A
authors.lastname: Spinhirne
authors.affiliation: Center for Systems Biology, University of Texas at Dallas, Richardson, TX 75080, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lai
authors.affiliation: Department of Molecular & Cell Biology, University of Texas at Dallas, Richardson, TX 75080, USA.
authors.forename: Samantha
authors.initials: S
authors.lastname: Preisser
authors.affiliation: Bioengineering Department, University of Texas at Dallas, Richardson, TX 75080, USA Electrical Engineering Department, University of Texas at Dallas, Richardson, TX 75080, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Bioengineering Department, University of Texas at Dallas, Richardson, TX 75080, USA Electrical Engineering Department, University of Texas at Dallas, Richardson, TX 75080, USA.
authors.forename: Taek
authors.initials: T
authors.lastname: Kang
authors.affiliation: Bioengineering Department, University of Texas at Dallas, Richardson, TX 75080, USA Department of Chemistry, University of Texas at Dallas, Richardson, TX 75080, USA Electrical Engineering Department, University of Texas at Dallas, Richardson, TX 75080, USA bleris@utdallas.edu.
authors.forename: Leonidas
authors.initials: L
authors.lastname: Bleris
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D012313:RNA / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D064130:CRISPR-Associated Proteins; D012313:RNA; D003851:Deoxyribonucleases