Creation, genotyping, and viability of rag2^Δ/Δ, il2rga ^−/− immunocompromised zebrafish. (A) Schematic of the rag2 locus on chromosome 25, with CRISPR-Cas9 gRNA targets noted by red bars. PCR primers used for genotyping are noted in blue. Deletion mutant and WT-specific PCR fragments are 444 bp and 973 bp, respectively. (B) DNA sequences for gRNAs noted by bold lettering and juxtaposed to the target genomic sequence. PAM sequences are noted by blue lettering and predicted cut sites by red arrows. (C) QIAxcel gel image of amplified PCR fragments from WT, heterozygous rag2^Δ/+ (HT), and homozygous rag2^Δ/Δ (HO) fish. (D) Survival statistics for rag2^Δ/Δ, il2rga^−/− zebrafish used in general engraftment studies shown in Fig. 1 and Fig. S2 (89.0% survival); in assessing immunotherapy responses following IP engraftment and injection of T cell products (94.9% survival); and in quantitating immune cell function at single-cell resolution following engraftment into the periocular musculature, IP injection with T cell products, and serial confocal imaging (93.3% survival). Animals largely died due to handling during imaging procedures associated with anesthesia, with none succumbing to overt infection during experiments outlined in this work (n = 984 animals).