![Figure 1. The somatic CRISPR–Cas9 technique generates conditional mutations in IFT components in C. elegans ciliated sensory neurons. (A) Schematic illustrating the use of somatic CRISPR–Cas9 for creating conditional mutants. (B) osm-3 and xbx-1 gene models. Exons are in blue, and arrows indicate sgRNA sequences corresponding to exons (Table S4). The NGG (PAM) sequences are denoted in red. (C) Representative gels showing the T7EI assay results for osm-3 and xbx-1. Indels are indicated at the bottom. (D, left) Schematic of cilia in WT and osm-3 animals. DS, distal segments; MS, middle segments; TZ, transition zone. (D, right) The morphology of amphid (top) and phasmid (bottom) cilia visualized using OSM-6::GFP. Arrowheads indicate junctions between the middle and distal segments. The length of the cilia are 8.2 ± 0.1 µm (amphids [A]) or 7.9 ± 0.3 µm (phasmids [P]) in WT, 4.8 ± 0.1 µm (A) or 3.9 ± 0.2 (P) µm in Phsp::Cas9; osm-3-sg, and 4.5 ± 0.1 µm (A) or 3.5 ± 0.1 µm (P) in Pdyf-1::Cas9; osm-3-sg. n = 23–81. Mean ± SE. Bar, 5 µm. (E) Dye-filling defects under a 100× objective lens. 0–4 and their respective colors represent the number of PHA/B neurons defective in dye-filling assays. n = 26–51. (F) Dye-filling defects under a fluorescence stereoscope. n = 150–315. Mean ± SE (error bars). (G) Kymographs (M or D) and corresponding lines (M’ or D’) of OSM-6::GFP motility. Error bars indicate mean ± SE. Horizontal bar, 2 µm; vertical bar, 5 s. n = 120–323. (H) Cilium morphology in conditional mutants visualized by OSM-6::GFP. Arrowheads indicate the positions where the junctions between the middle and distal segments should be in WT animals. Bar, 5 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/208/6/10.1083_jcb.201411041/6/jcb_201411041r_fig1.jpeg?Expires=1771560134&Signature=pM1SZNllaS2l3Lnl2VVb~-Y5NnCSxP1tAwHYrdlhxKq0NhC69aEfGxyikEme~GBD96eVhqxOmdkDK08MPVg39L0BJLFj-PxpbKuQDeCdAt75Jmfklt1Uy0Tkr9a2DWRIBxZT9k6fKzr4t0Qr6BVClqK3Jdy53f22UeocWv0VV74RqQlmt-q8ZioKtsu6JTYgdlRK-pi7FPDF6~ry~8L4WlH4FgcID6~H2RlE~PsGvqspm7eyJf19qSetNGYd3p0hcB0A928-D4GdAJOYzm9UaWOPdWrgc-CaaVOXnYkLraPOUsIye8NLEvp9KjVerHOphwYbLfS0ntQ2XxqiIJFKjg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The somatic CRISPR–Cas9 technique generates conditional mutations in IFT components in C. elegans ciliated sensory neurons. (A) Schematic illustrating the use of somatic CRISPR–Cas9 for creating conditional mutants. (B) osm-3 and xbx-1 gene models. Exons are in blue, and arrows indicate sgRNA sequences corresponding to exons (Table S4). The NGG (PAM) sequences are denoted in red. (C) Representative gels showing the T7EI assay results for osm-3 and xbx-1. Indels are indicated at the bottom. (D, left) Schematic of cilia in WT and osm-3 animals. DS, distal segments; MS, middle segments; TZ, transition zone. (D, right) The morphology of amphid (top) and phasmid (bottom) cilia visualized using OSM-6::GFP. Arrowheads indicate junctions between the middle and distal segments. The length of the cilia are 8.2 ± 0.1 µm (amphids [A]) or 7.9 ± 0.3 µm (phasmids [P]) in WT, 4.8 ± 0.1 µm (A) or 3.9 ± 0.2 (P) µm in Phsp::Cas9; osm-3-sg, and 4.5 ± 0.1 µm (A) or 3.5 ± 0.1 µm (P) in Pdyf-1::Cas9; osm-3-sg. n = 23–81. Mean ± SE. Bar, 5 µm. (E) Dye-filling defects under a 100× objective lens. 0–4 and their respective colors represent the number of PHA/B neurons defective in dye-filling assays. n = 26–51. (F) Dye-filling defects under a fluorescence stereoscope. n = 150–315. Mean ± SE (error bars). (G) Kymographs (M or D) and corresponding lines (M’ or D’) of OSM-6::GFP motility. Error bars indicate mean ± SE. Horizontal bar, 2 µm; vertical bar, 5 s. n = 120–323. (H) Cilium morphology in conditional mutants visualized by OSM-6::GFP. Arrowheads indicate the positions where the junctions between the middle and distal segments should be in WT animals. Bar, 5 µm.
