title: Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes.
abstract: Genome editing using the CRISPR/Cas9 system requires the presence of guide RNAs bound to the Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves the host cell genome at sites specified by the guide RNAs. New genetic material may be introduced during repair of the double-stranded break via homology dependent repair (HDR) if suitable DNA templates are delivered with the CRISPR components. Early methods used plasmid or viral vectors to make these components in the host cell, however newer approaches using recombinant Cas9 protein with synthetic guide RNAs introduced directly as an RNP complex into cells shows faster onset of action with fewer off-target effects. This approach also enables use of chemically modified synthetic guide RNAs that have improved nuclease stability and reduces the risk of triggering an innate immune response in the host cell. This article provides detailed methods for genome editing using the RNP approach with synthetic guide RNAs using lipofection or electroporation in mammalian cells or using microinjection in murine zygotes, with or without addition of a single-stranded HDR template DNA.
authors.affiliation: Integrated DNA Technologies, Inc., Coralville, IA 52241, USA.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Jacobi
authors.affiliation: Integrated DNA Technologies, Inc., Coralville, IA 52241, USA.
authors.forename: Garrett R
authors.initials: GR
authors.lastname: Rettig
authors.affiliation: Integrated DNA Technologies, Inc., Coralville, IA 52241, USA.
authors.forename: Rolf
authors.initials: R
authors.lastname: Turk
authors.affiliation: Integrated DNA Technologies, Inc., Coralville, IA 52241, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Collingwood
authors.affiliation: Integrated DNA Technologies, Inc., Coralville, IA 52241, USA.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Zeiner
authors.affiliation: Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE 68198, USA.
authors.forename: Rolen M
authors.initials: RM
authors.lastname: Quadros
authors.affiliation: Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE 68198, USA.
authors.forename: Donald W
authors.initials: DW
authors.lastname: Harms
authors.affiliation: Department of Neurobiology & Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Bonthuis
authors.affiliation: Department of Neurobiology & Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132, USA; Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Gregg
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa 259-1193, Japan; Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Kanagawa 259-1193, Japan; The Institute of Medical Sciences, Tokai University, Kanagawa 259-1193, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
authors.affiliation: Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE 68198, USA; Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: cgurumurthy@unmc.edu.
authors.forename: Channabasavaiah B
authors.initials: CB
authors.lastname: Gurumurthy
authors.affiliation: Integrated DNA Technologies, Inc., Coralville, IA 52241, USA. Electronic address: mbehlke@idtdna.com.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Behlke
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018274:Electroporation; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting / Q000379:methods; D018014:Gene Transfer Techniques; D016678:Genome; D057809:HEK293 Cells; D006801:Humans; D019169:Jurkat Cells; D008055:Lipids / Q000737:chemistry; D051379:Mice; D008845:Microinjections; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D059767:Recombinational DNA Repair; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D015053:Zygote / Q000166:cytology / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008055:Lipids; C086724:Lipofectamine; D000094704:RNA, Guide, CRISPR-Cas Systems; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR; Cas9; Genome editing; Homology directed repair (HDR); Ribonucleoprotein (RNP) complex; crRNA-tracrRNA