Extended Data Figure 9. Generation and clinical assessment of C57BL/6 mice with deletions in Ldlrad3 by CRISPR-Cas9 gene targeting.

a. Scheme of Ldlrad3 gene locus with two sgRNA targeting guides for a site in exon 2 of both isoforms. The full-length and truncated Δ32 N-terminus residue Ldlrad3 isoforms are colored red (top) and orange (bottom), respectively. b. Sequencing and alignment of Ldlrad3 sgRNA targeting region in exon 2 (11- and 14-nucleotide frameshift deletions) in gene-edited Ldlrad3 mice. The amino acid residues and the two sgRNA guides used for gene-editing (blue and orange arrows) are indicated above. c-d. Seven-week-old male and female mice with deletions in Ldlrad3 (Δ11 or Δ14 nucleotides; homozygous or compound heterozygous) or wild-type C57BL/6 mice were inoculated subcutaneously with 10³ PFU of VEEV TrD (c, left panel) or 10² FFU of VEEV ZPC738 (d). Mice were monitored for weight change. Data are from two experiments (VEEV TrD: WT, n = 12; ΔLdlrad3, n = 10; VEEV ZPC738: WT, n = 9; ΔLdlrad3, n = 8; two-way ANOVA with Dunnett’s post-test: ** P < 0.01, **** P < 0.0001; n.s., not significant). c: 1 dpi, P > 0.999; 2 dpi, P = 0.2136; 3 dpi, P = 0.5489; 4 dpi, P = 0.0065; 8 dpi, P = 0.0014. d: 1 dpi, P = 0.8383; 2 dpi, P = 0.001. Clinical disease (right panels) was assessed over time (healthy, ruffled fur, hunched/behavioral, seizures/ataxia, moribund, or death) in mice inoculated with VEEV TrD (c, right panels).