title: Cloning-free CRISPR.
abstract: We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%-4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.
authors.affiliation: Hubrecht Institute and UMC Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine at Utrecht University, Yalelaan 108, 3583 CM Utrecht, the Netherlands.
authors.forename: Mandana
authors.initials: M
authors.lastname: Arbab
authors.affiliation: Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA; Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
authors.forename: Sharanya
authors.initials: S
authors.lastname: Srinivasan
authors.affiliation: Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
authors.forename: Tatsunori
authors.initials: T
authors.lastname: Hashimoto
authors.affiliation: Hubrecht Institute and UMC Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine at Utrecht University, Yalelaan 108, 3583 CM Utrecht, the Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: Geijsen
authors.affiliation: Hubrecht Institute and UMC Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address: rsherwood@partners.org.
authors.forename: Richard I
authors.initials: RI
authors.lastname: Sherwood
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018390:Gene Targeting / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice