The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations.

Strategies for single base gene editing in an immortalized human cell line by CRISPR/Cas9 technology

Silvestri, Roberto;Dell'Anno, Irene;Romei, Cristina;Giovannoni, Roberto;Vitiello, Marianna;Cipollini, Monica;Elisei, Rossella;Landi, Stefano
Penultimo
;
Gemignani, Federica
Ultimo
2024-01-01

Abstract

The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations.
2024
Corrado, Alda; Aceto, Romina; Miglietta, Simona; Silvestri, Roberto; Dell'Anno, Irene; Lepori, Irene; Ricci, Benedetta; Romei, Cristina; Giovannoni, Roberto; Poliseno, Laura; Evangelista, Monica; Vitiello, Marianna; Cipollini, Monica; Elisei, Rossella; Landi, Stefano; Gemignani, Federica
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11568/1220768
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