CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells

STAR Protoc. 2021 Mar 24;2(2):100407. doi: 10.1016/j.xpro.2021.100407. eCollection 2021 Jun 18.


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).

Keywords: CRISPR; Cell Biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • Gene Editing
  • Gene Knock-In Techniques / methods*
  • Humans
  • Retinal Pigment Epithelium / cytology*
  • Ribonucleoproteins / genetics*


  • Ribonucleoproteins