CRISPR/Cas9-mediated site specific integration of LP onFer1L4
Fer1L4 single guide (sg) RNA sequence
(5΄-AGACGCCTAACAGAGCTGCCAGG-3΄) was designed using the CasFinder system
(Aach, Mali, & Church, 2014) and obtained
from ToolGen (Seoul, Korea). 2 × 106 cells were
transfected with 2 μg of LP DNA, 4 μg of Cas9 protein and 1 μg sgRNA
using SG cell line 4D-nucleofector® X Kit L (V4XC-3024; Lonza, Basel,
Switzerland). Transfected cells were selected for 2 weeks with 50 μM
blasticisdin (ant-bl-05; Invitrogen, Waltham, MA, USA). During
selection, the medium was changed every 4 d. After selection, single
cells were seeded per well in 150 μl medium in 96-well plates (353072;
Falcon) and incubated for 15 d. Cells expressing mCherry were screened
using fluorescence microscopy (Axiovert 200, Carl Zeiss, Oberkochen,
Germeny).