Generation of Flag-HA-GFP-DICER knock-in cell line by CRISPR/Cas9

To generate the knock-in cell line, the sequence of Flag-HA-GFP was amplified by PCR from the Flag-HA-GFP plasmid [85]. DNA sequences corresponding to 1 Kb upstream (left homology arm) and downstream (right homology arm) the starting codon (ATG) of DICER gene were amplified from HCT116 cell genomic DNA using primer pairs listed in S5 Table. The three PCR products were gel-purified and cloned into a linearized pUC19 by In-fusion cloning (Clontech) to obtain the template for homologous recombination (LarmDICER-FlagHAGFP-RarmDICER).

Design of the guide RNA targeting the region between Dicer 5’-UTR and its first coding exon for CRISPR/Cas9 mediated knock-in was carried out using the CRISPOR Design Tool [86]. Annealed oligonucleotides corresponding to the gRNA (S5 Table) were cloned into the vector pX459 (Addgene #48139) which also encodes S. pyogenes Cas9 with 2A-Puro.

The sequence of the donor plasmid was additionally mutagenized to disrupt the PAM sequence of the right homology arm to avoid its cleavage by the gRNA.

To obtain the knock-in (KI) cell line, 5 x 10⁵ HCT116 cells were seeded in a 6 well plate with Dulbecco’s modified Eagle medium (DMEM, Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Clontech) in a humidified atmosphere of 5% CO₂ at 37°C and transfected after 24 hours with the pX459-gRNADicerNterm-Cas9-2A-Puro plasmid and the Leftarm-FlagHAGFP-RightarmDICER donor plasmids at the ratio of 1 to 1 (6 micrograms plasmids in total) using Lipofectamine 2000 according to the manufacturer’s instructions. 24 hours later, puromycin (1 mg/mL) was added to the cells to increase the KI efficiency and genomic DNA was isolated from individual colonies few days later.

The presence of the Flag-HA-GFP tag in frame with hDICER coding sequence was confirmed by sequencing PCR amplicon from KI cell gDNA. Expression of Flag-HA-GFP N-terminal tagged Dicer protein in the KI cells was confirmed by western blot.