hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a protocol for rapid and efficient generation of diverse homozygous knockins. In contrast to other approaches, this strategy bypasses the need for molecular cloning. Our approach can also be applied to a variety of cell types including cancer and induced pluripotent stem cells (iPSCs).
CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells / Ghetti, S.; Burigotto, M.; Mattivi, A.; Magnani, G.; Casini, A.; Bianchi, A.; Cereseto, A.; Fava, L.. - In: STAR PROTOCOLS. - ISSN 2666-1667. - ELETTRONICO. - 2:2(2021), pp. 10040701-10040712. [10.1016/j.xpro.2021.100407]
CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells
Burigotto M.;Mattivi A.;Magnani G.;Casini A.;Cereseto A.;Fava L.
2021-01-01
Abstract
hTERT-RPE1 cells are genetically stable near diploid cells widely used to model cell division, DNA repair, or ciliogenesis in a non-transformed context. However, poor transfectability and limited homology-directed repair capacity hamper their amenability to gene editing. Here, we describe a protocol for rapid and efficient generation of diverse homozygous knockins. In contrast to other approaches, this strategy bypasses the need for molecular cloning. Our approach can also be applied to a variety of cell types including cancer and induced pluripotent stem cells (iPSCs).| File | Dimensione | Formato | |
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