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.

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]

Formation and nucleolytic processing of Cas9-induced DNA breaks in human cells quantified by droplet digital PCR

D. Dibitetto
Primo
Membro del Collaboration Group
;
F. Marini
Penultimo
Membro del Collaboration Group
;
A. Pellicioli
Ultimo
Funding Acquisition
2018

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.
No
English
DSB repair; DSB resection; Cas9; ddPCR; gene editing
Settore BIO/11 - Biologia Molecolare
Review essay
Esperti anonimi
Ricerca di base
Pubblicazione scientifica
   Insights into the functions of DNA damage processing and repair factors to design novel selective anticancer drugs.
   MINISTERO DELL'ISTRUZIONE E DEL MERITO
   2015LZE994_001
ago-2018
30-giu-2018
Elsevier
68
68
74
7
Pubblicato
Periodico con rilevanza internazionale
Aderisco
info:eu-repo/semantics/article
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]
partially_open
Prodotti della ricerca::01 - Articolo su periodico
5
262
Article (author)
no
D. Dibitetto, M. La Monica, M. Ferrari, F. Marini, A. Pellicioli
File in questo prodotto:
File Dimensione Formato  
pagination_DNAREP_2496 19.05.38.pdf

accesso aperto

Tipologia: Pre-print (manoscritto inviato all'editore)
Dimensione 1.42 MB
Formato Adobe PDF
1.42 MB Adobe PDF Visualizza/Apri
1-s2.0-S1568786418300260-main.pdf

accesso riservato

Tipologia: Publisher's version/PDF
Dimensione 1.49 MB
Formato Adobe PDF
1.49 MB Adobe PDF   Visualizza/Apri   Richiedi una copia
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/582482
Citazioni
  • ???jsp.display-item.citation.pmc??? 4
  • Scopus 10
  • ???jsp.display-item.citation.isi??? 10
  • OpenAlex ND
social impact