{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,27]],"date-time":"2026-10-27T06:52:33Z","timestamp":1793083953308,"version":"4.5.0"},"reference-count":26,"publisher":"eLife Sciences Publications, Ltd","license":[{"start":{"date-parts":[[2014,12,15]],"date-time":"2014-12-15T00:00:00Z","timestamp":1418601600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2014,12,15]],"date-time":"2014-12-15T00:00:00Z","timestamp":1418601600000},"content-version":"am","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2014,12,15]],"date-time":"2014-12-15T00:00:00Z","timestamp":1418601600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100001021","name":"Damon Runyon Cancer Research Foundation (Damon Runyon)","doi-asserted-by":"publisher","award":["DRG2176-13"],"award-info":[{"award-number":["DRG2176-13"]}],"id":[{"id":"10.13039\/100001021","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100004337","name":"Roche","doi-asserted-by":"publisher","award":["Postdoctoral Fellowship, RPF311"],"award-info":[{"award-number":["Postdoctoral Fellowship, RPF311"]}],"id":[{"id":"10.13039\/100004337","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"abstract":"<jats:p>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 (&lt;xref ref-type=\"bibr\" rid=\"bib13\"&gt;Jinek et al., 2013&lt;\/xref&gt;), 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.<\/jats:p>","DOI":"10.7554\/elife.04766","type":"journal-article","created":{"date-parts":[[2014,12,15]],"date-time":"2014-12-15T07:32:50Z","timestamp":1418628770000},"source":"Crossref","is-referenced-by-count":1094,"title":["Enhanced homology-directed human genome engineering by controlled timing of CRISPR\/Cas9 delivery"],"prefix":"10.7554","volume":"3","author":[{"given":"Steven","family":"Lin","sequence":"first","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brett T","family":"Staahl","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ravi K","family":"Alla","sequence":"additional","affiliation":[{"name":"Computational Genomics Resource Laboratory, QB3, University of California, Berkeley, Berkeley, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer A","family":"Doudna","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States"},{"name":"Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States"},{"name":"Department of Chemistry, University of California, Berkeley, Berkeley, United States"},{"name":"Department of Chemistry, Lawrence Berkeley National Laboratory, Berkeley, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"4374","published-online":{"date-parts":[[2014,12,15]]},"reference":[{"key":"bib1","doi-asserted-by":"crossref","first-page":"1314","DOI":"10.1016\/S0021-9258(18)96181-8","article-title":"Hydroxyurea reversal of inhibition and use as a cell-synchronizing agent","volume":"242","author":"Adams","year":"1967","journal-title":"The Journal of Biological Chemistry"},{"key":"bib2","doi-asserted-by":"publisher","first-page":"595","DOI":"10.1038\/nature04678","article-title":"NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21","volume":"441","author":"Arron","year":"2006","journal-title":"Nature"},{"key":"bib3","doi-asserted-by":"publisher","first-page":"1479","DOI":"10.1016\/j.cell.2013.12.001","article-title":"Dynamic imaging of genomic lociin living human cells by an optimized CRISPR\/Cas system","volume":"155","author":"Chen","year":"2013","journal-title":"Cell"},{"key":"bib4","doi-asserted-by":"publisher","first-page":"819","DOI":"10.1126\/science.1231143","article-title":"Multiplex genome engineering using CRISPR\/Cas systems","volume":"339","author":"Cong","year":"2013","journal-title":"Science"},{"key":"bib5","doi-asserted-by":"publisher","first-page":"872","DOI":"10.1126\/science.272.5263.872","article-title":"HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor","volume":"272","author":"Feng","year":"1996","journal-title":"Science"},{"key":"bib6","doi-asserted-by":"publisher","first-page":"6636","DOI":"10.1128\/MCB.22.18.6636-6647.2002","article-title":"Dyrk1A haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice","volume":"22","author":"Fotaki","year":"2002","journal-title":"Molecular and Cellular Biology"},{"key":"bib7","doi-asserted-by":"publisher","first-page":"822","DOI":"10.1038\/nbt.2623","article-title":"High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells","volume":"31","author":"Fu","year":"2013","journal-title":"Nature Biotechnology"},{"key":"bib8","first-page":"157","article-title":"Synchronization of Cell Populations in G1\/S and G2\/M Phases of the Cell Cycle","volume-title":"Methods in Molecular Biology","volume":"296","author":"Harper","year":"2007"},{"key":"bib9","doi-asserted-by":"publisher","first-page":"113","DOI":"10.1146\/annurev-genet-051710-150955","article-title":"Regulation of homologous recombination in eukaryotes","volume":"44","author":"Heyer","year":"2010","journal-title":"Annual Review of Genetics"},{"key":"bib10","doi-asserted-by":"publisher","first-page":"827","DOI":"10.1038\/nbt.2647","article-title":"DNA targeting specificity of RNA-guided Cas9 nucleases","volume":"31","author":"Hsu","year":"2013","journal-title":"Nature Biotechnology"},{"key":"bib11","doi-asserted-by":"publisher","first-page":"264","DOI":"10.3109\/10409238.2012.658112","article-title":"Targeted gene therapies: tools, applications, optimization","volume":"47","author":"Humbert","year":"2012","journal-title":"Critical Reviews in Biochemistry and Molecular Biology"},{"key":"bib12","doi-asserted-by":"publisher","DOI":"10.1002\/0471143030.cb0803s00","article-title":"Methods for synchronizing cells at specific stages of the cell cycle","author":"Jackman","year":"2001","journal-title":"Current Protocols in Cell Biology"},{"key":"bib12a","doi-asserted-by":"publisher","first-page":"816","DOI":"10.1126\/science.1225829","article-title":"A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity","volume":"337","author":"Jinek","year":"2012","journal-title":"Science"},{"key":"bib13","doi-asserted-by":"publisher","first-page":"e00471","DOI":"10.7554\/eLife.00471","article-title":"RNA-programmed genome editing in human cells","volume":"2","author":"Jinek","year":"2013","journal-title":"eLife"},{"key":"bib14","doi-asserted-by":"publisher","first-page":"1012","DOI":"10.1101\/gr.171322.113","article-title":"Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins","volume":"24","author":"Kim","year":"2014","journal-title":"Genome Research"},{"key":"bib15","doi-asserted-by":"publisher","first-page":"833","DOI":"10.1038\/nbt.2675","article-title":"CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering","volume":"31","author":"Mali","year":"2013a","journal-title":"Nature Biotechnology"},{"key":"bib16","doi-asserted-by":"publisher","first-page":"823","DOI":"10.1126\/science.1232033","article-title":"RNA-Guided human genome engineering via Cas9","volume":"339","author":"Mali","year":"2013b","journal-title":"Science"},{"key":"bib17","doi-asserted-by":"publisher","first-page":"833","DOI":"10.1056\/NEJM200109133451113","article-title":"Chemokines and the molecular basis of cancer metastasis","volume":"345","author":"Murphy","year":"2001","journal-title":"The New England Journal of Medicine"},{"key":"bib18","doi-asserted-by":"publisher","first-page":"246","DOI":"10.1038\/nature10989","article-title":"Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations","volume":"485","author":"O'Roak","year":"2012","journal-title":"Nature"},{"key":"bib19","doi-asserted-by":"publisher","first-page":"189","DOI":"10.1126\/science.1248024","article-title":"Mitosis inhibits DNA double-strand break repair to guard against telomere fusions","volume":"344","author":"Orthwein","year":"2014","journal-title":"Science"},{"key":"bib20","doi-asserted-by":"publisher","first-page":"839","DOI":"10.1038\/nbt.2673","article-title":"High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity","volume":"31","author":"Pattanayak","year":"2013","journal-title":"Nature Biotechnology"},{"key":"bib21","doi-asserted-by":"publisher","first-page":"135","DOI":"10.1016\/j.cell.2013.08.031","article-title":"The cell-cycle state of stem cells determines cell fate propensity","volume":"155","author":"Pauklin","year":"2013","journal-title":"Cell"},{"key":"bib22","doi-asserted-by":"publisher","first-page":"1380","DOI":"10.1016\/j.cell.2013.08.021","article-title":"Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity","volume":"154","author":"Ran","year":"2013","journal-title":"Cell"},{"key":"bib23","doi-asserted-by":"publisher","first-page":"e96483","DOI":"10.1371\/journal.pone.0096483","article-title":"The position of DNA cleavage by TALENs and cell synchronization influences the frequency of gene editing directed by single-stranded oligonucleotides","volume":"9","author":"Rivera-Torres","year":"2014","journal-title":"PLOS ONE"},{"key":"bib24","doi-asserted-by":"publisher","first-page":"134","DOI":"10.1038\/cr.2007.111","article-title":"Regulation of DNA double-strand break repair pathway choice","volume":"18","author":"Shrivastav","year":"2008","journal-title":"Cell Research"},{"key":"bib24a","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3081","article-title":"Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo","author":"Zuris","year":"2014","journal-title":"Nat. Biotechnol"}],"container-title":["eLife"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/elifesciences.org\/lookup\/doi\/10.7554\/eLife.04766","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/cdn.elifesciences.org\/articles\/04766\/elife-04766-v2.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/cdn.elifesciences.org\/articles\/04766\/elife-04766-v2.xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/elifesciences.org\/articles\/04766","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,10,11]],"date-time":"2023-10-11T20:49:05Z","timestamp":1697057345000},"score":1,"resource":{"primary":{"URL":"https:\/\/elifesciences.org\/articles\/04766"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,12,15]]},"references-count":26,"alternative-id":["10.7554\/eLife.04766"],"URL":"https:\/\/doi.org\/10.7554\/elife.04766","archive":["CLOCKSS"],"relation":{"has-review":[{"id-type":"doi","id":"10.3410\/f.725276483.793511758","asserted-by":"object"},{"id-type":"doi","id":"10.7554\/eLife.04766.010","asserted-by":"object"},{"id-type":"doi","id":"10.7554\/eLife.04766.009","asserted-by":"object"}]},"ISSN":["2050-084X"],"issn-type":[{"value":"2050-084X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,12,15]]},"article-number":"e04766"}}