RNA-programmed genome editing in human cells

By Martin Jinek, Alexandra East, Aaron T. Cheng, Steven Lin, E. Ma, Jennifer A. Doudna. eLife, 2013

2,073 citations recorded by the source corpus.

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells. DOI: http://dx.doi.org/10.7554/eLife.00471.001

Subfields: Epidemiology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/c9f8479a8298afdab168a43405c60cd465ee64c2

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Citations in the corpus

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila (2014, methodology): “pyogenes Cas9 [codon optimized for expression in human (13, 14) or Drosophila cells] fused to”
Development and Applications of CRISPR-Cas9 for Genome Engineering (2014, positive): “SpCas9 can be targeted either with a pair of crRNA and tracrRNA (Cong et al., 2013) or with a chimeric sgRNA (Cong et al., 2013; Mali et al., 2013a; Cho et al., 2013; Jinek et al., 2013), as the crRNA or sgRNA contains a 20 nt guide sequence that directly matches the target site.”
Molecular targeted therapy: Treating cancer with specificity (2018, background): “This was further investigated in human cells (Jinek et al., 2013) using a single-guide RNA (sgRNA) libraries.”
Concerning RNA-guided gene drives for the alteration of wild populations (2014, background): “Genome editing was democratized by the discovery and adaptation of Cas9, an enzyme that can be programmed to cut target DNA sequences specified by a guiding RNA molecule (Deltcheva et al., 2011; Jinek et al., 2012; Cho et al., 2013; Cong et al., 2013; Jinek et al., 2013; Mali et al., 2013c).”