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Unexpected heterogeneity derived from Cas9 ribonucleoprotein‐introduced clonal cells at the HPRT 1 locus
Author(s) -
Sakuma Tetsushi,
Mochida Keiji,
Nakade Shota,
Ezure Toru,
Minagawa Sachi,
Yamamoto Takashi
Publication year - 2018
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/gtc.12569
Subject(s) - biology , transfection , cas9 , crispr , cloning (programming) , microbiology and biotechnology , genome editing , ribonucleoprotein , sanger sequencing , genetics , cell culture , gene , mutation , rna , computer science , programming language
Single‐cell cloning is an essential technique for establishing genome‐edited cell clones mediated by programmable nucleases such as CRISPR ‐Cas9. However, residual genome‐editing activity after single‐cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR ‐Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins ( RNP s). In this study, we investigated how the timing for single‐cell cloning of Cas9 RNP ‐transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNP s targeting several loci in the HPRT 1 gene in HCT 116 cells, followed by single‐cell cloning at 24, 48, 72 hr and 1 week post‐transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high‐resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long‐term incubation before single‐cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNP s were not removed before single‐cell cloning. Therefore, the active Cas9 RNP s were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNP s may cause continuous introduction of gene mutations depending on the experimental procedures.