title: Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.
abstract: The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
authors.forename: Steven
authors.initials: S
authors.lastname: Lin
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
authors.forename: Brett T
authors.initials: BT
authors.lastname: Staahl
authors.affiliation: Computational Genomics Resource Laboratory, QB3, University of California, Berkeley, Berkeley, United States.
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Alla
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics; D002470:Cell Survival; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004622:Embryo, Mammalian; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D059767:Recombinational DNA Repair; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D013210:Staphylococcus / Q000737:chemistry / Q000201:enzymology; D013997:Time Factors
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA; D004720:Endonucleases
keywords: CRISPR/Cas9; cell biology; cell cycle synchronization; chromosomes; genes; genome engineering; homologous recombination; human; nocodazole; non-homologous end joining