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

By Steven Lin, Brett T. Staahl, R. Alla, Jennifer A. Doudna. eLife, 2014

1,111 citations recorded by the source corpus.

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. DOI: http://dx.doi.org/10.7554/eLife.04766.001

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/97714033fa26b814d710b6e60d3104d8bd59c98c

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Citations in the corpus

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects (2020, methodology): “Moreover, the CCR5-specific TALEN was able to distinguish between the CCR5 target locus and a highly similar site in CCR2 when compared with ZFN technology.37 By adopting TALEN gene editing technology, precise disruptions have also been introduced into the T cell receptor α constant (TRAC) gene and ”
OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization (2022, background): “Moreover, various DNA repair mechanisms compete with homologous recombination for the resolution of CRISPR-induced genomic breaks ( 25 ) so that alleles containing nonfunctional mutations can be present inaddition to thedesiredfusionalleles.”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “Compared with vector-mediated nucleotide delivery methods, Cas9-mediated genome editing via RNP delivery methods (108, 161, 253, 257, 258) exhibits higher fidelity and lower cell toxicity, bypasses the safety problems of introducing foreign DNA into the host genome and, thus, may provide a platform ”
Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA[OPEN] (2015, background): “Furthermore, transient delivery of purified Cas9 protein and gRNA (as a ribonucleoprotein complex) into human culture cells has shown high frequencies of targeted mutagenesis and gene editing while reducing off-target mutations (Kim et al., 2014; Lin et al., 2014).”
Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes (2016, positive): “DNA-free genome modiﬁcation was ﬁrst demonstrated in mammalian cell cultures 12,13 .”