Generation of B16-F1 Lpd KO cell lines

Generation of B16-F1 mouse melanoma Lpd KO clones was performed analogous to the method used for generating FMNL2/3 KO clones described previously (Kage et al., 2017). CRISPR/Cas9 guide design was performed using a publicly available CRISPR design tool (then http://crispr.mit.edu), with the DNA sequence of exon 5 of Lpd/Raph1 being used [base pairs 937–1014 of complementary DNA (cDNA) of {"type":"entrez-nucleotide","attrs":{"text":"NM_001045513.3","term_id":"341604761","term_text":"NM_001045513.3"}}NM_001045513.3]. A guide with the highest available aggregate score of 75/100 was selected, targeting the following genomic DNA sequence: 5′-TGAGAAGATCCGAGTTGCTC-3′. Forward and reverse guide oligonucleotide sequences, 5′-CACCGTGAGAAGATCCGAGTTGCTC-3′ and 5′-AAACGAGCAACTCGGATCTTCTCAC-3′, respectively, were annealed for 4 min at 95°C, followed by 10 min incubation at 70°C and gradual cooling at room temperature in a buffer containing 100 mM KAc, 2 mM MgAc, 30 mM HEPES-KOH (pH 4.7). Annealed sequences were cloned into an expression vector pSpCas9(BB)-2A-Puro(px459) (Addgene plasmid #48138) using BbsI restriction enzyme. Successful cloning was confirmed by sequencing with primer 5′-AGGCTGTTAGAGAGATAATTGG-3′. For generation of Lpd KO cell clones, B16-F1 cells were transfected with the cloned plasmid encoding puromycin resistance and the CRISPR guide sequence targeting Lpd. Transfected cells were cultured for 4 days with B16-F1 culture medium containing 2.5 mg/ml puromycin. Single colonies were isolated and expanded until confluent. Confirmation of genetic knockout was validated by both western blotting and DNA sequencing of genomic DNA. For guide DNA extraction, B16-F1 cells were pelleted and incubated at 55°C overnight in lysis buffer (100 mM Tris-HCl pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl) containing 40 µg/ml proteinase K. Standard phenol/chloroform precipitation was performed for extraction of nucleic acids. Genomic DNA sequence of 364 bp, flanking the Lpd target sequence, was amplified by Phusion High-Fidelity Polymerase (New England Biolabs) with 5′-GAACGGGCCATTTTAAAATTGTGC-3′ and 5′-AGACATTAGGAAGAATACAGTTTTACC-3′ as forward and reverse primers, respectively. Amplified sequence was purified with NucleoSpin Gel and a PCR clean-up kit according to the manufacturer's instructions (Macherey&Nagel), cloned into a Zero Blunt TOPO vector using Zero Blunt TOPO Cloning Kit (Invitrogen) and transformed into competent bacteria. Single bacterial colonies were isolated and inoculated, plasmid DNA purified using a NucleoSpin Plasmid kit (Macherey&Nagel) and sequenced by MWG-Biotech (Ebersberg, Germany) using sequencing primer 5′-CAGGAAACAGCTATGAC-3′. Clones with frameshift mutations on all alleles causing stop codons downstream of the target site were selected for further characterization.