Targeted Gene Activation Using RNA-Guided Nucleases.
The discovery of the prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system and its adaptation for targeted manipulation of DNA in diverse species has revolutionized the field of genome engineering. In particular, the fusion of catalytically inactive Cas9 to any number of transcriptional activator domains has resulted in an array of easily customizable synthetic transcription factors that are capable of achieving robust, specific, and tunable activation of target gene expression within a wide variety of tissues and cells. This chapter describes key experimental design considerations, methods for plasmid construction, gene delivery protocols, and procedures for analysis of targeted gene activation in mammalian cell lines using CRISPR-Cas transcription factors.
Department of Bioengineering, University of Illinois at Urbana-Champaign, 1270 Digital Computer Laboratory, MC-278, 1304 West Springfield Avenue, Urbana, IL, 61801-2910, USA.
Alexander
A
Brown
Department of Bioengineering, University of Illinois at Urbana-Champaign, 1270 Digital Computer Laboratory, MC-278, 1304 West Springfield Avenue, Urbana, IL, 61801-2910, USA.
Wendy S
WS
Woods
Department of Bioengineering, University of Illinois at Urbana-Champaign, 1270 Digital Computer Laboratory, MC-278, 1304 West Springfield Avenue, Urbana, IL, 61801-2910, USA. pablo@illinois.edu.
Pablo
P
Perez-Pinera
D064113:CRISPR-Cas Systems; D000072669:Gene Editing; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D015533:Transcriptional Activation
D000094704:RNA, Guide, CRISPR-Cas Systems; D012260:Ribonucleases
CRISPR-Cas9; Gene activation; Gene expression; Genome engineering; RNA-guided nucleases; Synthetic biology; Transcription