title: Multiplexed Targeted Genome Engineering Using a Universal Nuclease-Assisted Vector Integration System.
abstract: Engineered nucleases are capable of efficiently modifying complex genomes through introduction of targeted double-strand breaks. However, mammalian genome engineering remains limited by low efficiency of heterologous DNA integration at target sites, which is typically performed through homologous recombination, a complex, ineffective and costly process. In this study, we developed a multiplexable and universal nuclease-assisted vector integration system for rapid generation of gene knock outs using selection that does not require customized targeting vectors, thereby minimizing the cost and time frame needed for gene editing. Importantly, this system is capable of remodeling native mammalian genomes through integration of DNA, up to 50 kb, enabling rapid generation and screening of multigene knockouts from a single transfection. These results support that nuclease assisted vector integration is a robust tool for genome-scale gene editing that will facilitate diverse applications in synthetic biology and gene therapy.
authors.affiliation: Department of Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Alexander
authors.initials: A
authors.lastname: Brown
authors.affiliation: Department of Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Wendy S
authors.initials: WS
authors.lastname: Woods
authors.affiliation: Department of Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Pablo
authors.initials: P
authors.lastname: Perez-Pinera
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D051356:Cortactin / Q000235:genetics; D004720:Endonucleases / Q000235:genetics; D000072669:Gene Editing; D055786:Gene Knockout Techniques / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D016678:Genome; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D045325:HCT116 Cells; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems; D000069896:Transcription Activator-Like Effector Nucleases; D014162:Transfection; D019076:Transgenes
chemical_list: D001426:Bacterial Proteins; C495868:CTTN protein, human; D051356:Cortactin; D000094704:RNA, Guide, CRISPR-Cas Systems; D049452:Green Fluorescent Proteins; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D000069896:Transcription Activator-Like Effector Nucleases
keywords: CRISPR; DNA recombination; TALEN; gene editing; genome engineering; synthetic biology; targeted genome integration