Formation and nucleolytic processing of Cas9-induced DNA breaks in human cells quantified by droplet digital PCR
Articolo
Data di Pubblicazione:
2018
Citazione:
Formation and nucleolytic processing of Cas9-induced DNA breaks in human cells quantified by droplet digital PCR / D. Dibitetto, M. La Monica, M. Ferrari, F. Marini, A. Pellicioli. - In: DNA REPAIR. - ISSN 1568-7864. - 68(2018 Aug), pp. 68-74. [10.1016/j.dnarep.2018.06.005]
Abstract:
Cas9 endonuclease from S. pyogenes is widely used to induce controlled double strand breaks (DSB) at desired
genomic loci for gene editing. Here, we describe a droplet digital PCR (ddPCR) method to precisely quantify the
kinetic of formation and 5′-end nucleolytic processing of Cas9-induced DSB in different human cells lines.
Notably, DSB processing is a finely regulated process, which dictates the choice between non-homologous end
joining (NHEJ) and homology directed repair (HDR). This step of DSB repair is also a relevant point to be taken
into consideration to improve Cas9-mediated technology. Indeed, by this protocol, we show that processing of
Cas9-induced DSB is impaired by CTIP or BRCA1 depletion, while it is accelerated after down-regulation of DNAPKcs
and 53BP1, two DSB repair key factors. In conclusion, the method we describe here can be used to study
DSB repair mechanisms, with direct utility for molecularly optimising the knock-out/in outcomes in genome
manipulation.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
DSB repair; DSB resection; Cas9; ddPCR; gene editing
Elenco autori:
D. Dibitetto, M. La Monica, M. Ferrari, F. Marini, A. Pellicioli
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