Abstract
BackgroundThe ability to modify the genome of any cell at a precise location has drastically improved with the recent discovery and implementation of CRISPR/Cas9 editing technology. However, the capacity to introduce specific directed changes at given loci is hampered by the fact that the major cellular repair pathway that occurs following Cas9-mediated DNA cleavage is the erroneous non-homologous end joining (NHEJ) pathway. Homology-directed recombination (HDR) is far less efficient than NHEJ and makes screening of clones containing directed changes time-consuming and labor-intensive.MethodsWe investigated the possibility of pharmacologically inhibiting DNA-PKcs, a key player in NHEJ, using small molecule inhibitors (NU7441 and KU-0060648), to ameliorate the rates of HDR repair events. These compounds were tested in a sensitive reporter assay capable of simultaneously informing on NHEJ and HDR, as well as on an endogenous gene targeted by Cas9.ResultsWe find that NU7441 and KU-0060648 reduce the frequency of NHEJ while increasing the rate of HDR following Cas9-mediated DNA cleavage.ConclusionsOur results identify two small molecules compatible for use with Cas9-editing technology to improve the frequency of HDR.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0215-6) contains supplementary material, which is available to authorized users.
Highlights
The ability to modify the genome of any cell at a precise location has drastically improved with the recent discovery and implementation of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing technology
We identified two small molecules compatible for use with Cas9-editing technology that improve the frequency of Homologydirected recombination (HDR) while reducing non-homologous end joining (NHEJ) mutagenic events
For γ-irradiation, 293/Traffic Light Reporter’ (TLR) cells were plated at 25 % confluency and the day were treated with DNA-PK inhibitors (2 μM NU7441 or 250 nM KU-0060648) for 1 h followed by 4 A Gray unit of ionization (GY) of γ-irradiation
Summary
The ability to modify the genome of any cell at a precise location has drastically improved with the recent discovery and implementation of CRISPR/Cas editing technology. Cas will generate double-stranded breaks (DSB) at the target site which are repaired by the erroneous non-homologous end-joining (NHEJ) pathway. NHEJ is initiated by a DSB leading to recruitment of a Ku70/80 heterodimer and the catalytic subunit of DNAdependent protein kinase (DNA-PKcs). This tethers the two ends and a series of subsequent reactions result in resection, extension, and ligation [8]. NHEJ is the more frequently observed repair pathway when using Cas9-mediated genome engineering, even in the presence of a donor HDR template [9,10,11,12]. Identifying cells of interest following HDR-directed editing is a labor-intensive and time-consuming process involving screening of individual clones to identify appropriate, correctly modified cells
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