Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

• Published in: eLife Vol. 3; p. e04766
• Main Authors: Lin, Steven, Staahl, Brett T, Alla, Ravi K, Doudna, Jennifer A
• Format: Journal Article
• Language: English
• Published: England eLife Sciences Publications Ltd 15.12.2014; eLife Sciences Publications, Ltd
• Subjects: Base Sequence; Cell Biology; Cell cycle; Cell Cycle - genetics; cell cycle synchronization; Cell Survival; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR/Cas9; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA Breaks, Double-Stranded; DNA damage; Embryo cells; Embryo fibroblasts; Embryo, Mammalian; Embryonic Stem Cells - cytology; Embryonic Stem Cells - metabolism; Endonucleases - genetics; Endonucleases - metabolism; Engineering; Fibroblasts - cytology; Fibroblasts - metabolism; Gene Expression; Gene Transfer Techniques; Genes and Chromosomes; Genetic Engineering - methods; Genetic testing; Genome editing; genome engineering; Genome, Human; Genomes; HEK293 Cells; homologous recombination; Homology; Humans; Infant, Newborn; Laboratories; Life Sciences & Biomedicine - Other Topics; Molecular Sequence Data; Neonates; nocodazole; non-homologous end joining; Recombinational DNA Repair; Research Advance; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - chemistry; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Sequence Analysis, DNA; Signal Transduction; Staphylococcus - chemistry; Staphylococcus - enzymology; Stem cell transplantation; Stem cells; Time Factors; United States > US; California; cell biology; CRISPR/Cas9; homologous recombination; genes; chromosomes; non-homologous end joining; cell cycle synchronization; human; genome engineering; nocodazole
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/elife.04766

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.