WAVE2ΔVCA localizes to the leading edge of sheet-like protrusions. (A) Generation of WAVE2ΔVCA cells with the split-GFP endogenous knock-in CRISPR/Cas9 editing system. Schematic of WAVE2 domains and editing (SHD, scar homology domain; BD, basic domain; PRD, proline-rich domain; V/WH2, WASP-homology 2 domain; CA, central acidic domain). In a GFP1-10–expressing cell line, CRISPR/Cas9 with a guide targeting WAVE2’s VCA region cuts and the cell repairs with a homology-directed repair (HDR) donor containing GFP11 and two stop codons. After generating clonal cell lines, the edited region was PCR-amplified and analyzed with MiSeq and CRISPresso2. The edited alleles are one HDR allele, one mixed HDR/NHEJ allele (which resulted in a stop codon), and one allele with a deletion >400 bp. (B) Western blot of WAVE2 and GAPDH in wild type and WAVE2ΔVCA GFP11 knock-in HEK-293T lines. (C) WAVE2ΔVCA localizes to the leading edge of sheet-like protrusions. Time lapse of endogenously split-GFP–edited full-length WAVE2 (FL KI, top row) and WAVE2ΔVCA (ΔVCA KI, bottom row) HEK-293T cells stimulated with 300 nM PMA. TIRF imaging; scale bar: 10 µm. See Video 10.