title: Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA.
abstract: While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA. In addition, the use of a short double stranded DNA oligonucleotide with 3' overhangs allowed integration of a longer FLAG epitope tag along with a restriction site at rates of up to 50%. We propose a model that favors the design of donor DNAs with the change as close to the cleavage site as possible. For small changes such as SNPs or short insertions, asymmetric single stranded donor molecules with 30 base homology arms 3' to the insertion/repair cassette and greater than 40 bases of homology on the 5' end seems to be favored. For larger insertions such as an epitope tag, a dsDNA donor with protruding 3' homology arms of 30 bases is favored. In both cases, protecting the ends of the donor DNA with phosphorothioate modifications improves the editing efficiency.
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Xiquan
authors.initials: X
authors.lastname: Liang
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA. Electronic address: jason.potter@thermofisher.com.
authors.forename: Jason
authors.initials: J
authors.lastname: Potter
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Shantanu
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Namritha
authors.initials: N
authors.lastname: Ravinder
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D055879:Gene Knock-In Techniques; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D059765:Homologous Recombination / Q000235:genetics*; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR; Cas9; Genome editing; Homologous recombination; Knock-in; gRNA