4.5. Cas9 and sgRNA Constructs

We used plasmids encoding Cas9-2A-mRFP-2A-PAC (puromycin N-acetyl-transferase, puromycin resistance gene) and single-guide RNAs (sgRNAs) purchased from Toolgen (Seoul, South Korea). The sgRNA target sequences were designed on the basis of bioinformatics tools (www.broadinstitute.org) and cloned into the vectors as described previously [40]. Briefly, oligonucleotides containing each target sequence were synthesized (Bioneer, Seoul, South Korea), and T4 polynucleotide kinase was used to add terminal phosphates to the annealed oligonucleotides (Biorad, CA, USA). Vector was digested with BsaI and ligated the annealed oligonucleotides into the vector. Oligonucleotide sequences are listed in Table 1.

Oligonucleotides used in this study.