Figure 1.

Generation and characterization of a casp^K551R transgenic fly by CRISPR/Cas9 genome editing.

(A) Casp is a negative regulator of IMD/NFkB signaling. Casp negatively regulates DREDD activity. DREDD is the protease responsible for cleavage/maturation of Relish (Rel), in response to IMD signaling and leads to the subsequent release of its N-terminal fragment Rel68, which travels to the nucleus to activate transcription. (B) Domain structure of Casp. Casp is orthologous to mammalian Fas associated factor 1 (FAF1) and contains an N-terminal Ubiquitin interaction domain (UBA), a ubiquitin-like (UAS) self-association domain and a C-terminal Ubiquitin regulatory domain (UBX). UBA domains are known to interact with Ubiquitin (Ub) and polyUb-chains (connected circles) while UBX binds specifically to the Transitional-ER-ATPase 94 (TER94; also known as valsolin containing protein -VCP or p97), which is member of the degradative Ub-proteasomal recycling system (Tresse et al., 2010). The site of Casp SUMOylation (K551) (Handu et. al., 2015) is marked in red on the figure. Double-headed arrows indicate non-covalent interactions between Casp and other proteins (C) Generation of putative lines for screening. 350 0-3 hour old embryos expressing Act5c-Cas9 along with the gRNA were injected with a 100 bp donor single-stranded oligonucleotide (ssODN). Adult males (n=123, one male per vial) that developed from these embryos were crossed to II^nd chromosome balancer females as a first step to stabilize putative mutant lines. Next, individual single parent crosses were set up to generate homozygous lines that were used to screen for mutants. (D) Screening for the casp^K551R mutant. PCR primers were designed to amplify 566 bp region spanning the site of mutation within the casp locus. A unique BssHII restriction site was engineered in the ssODN, allowing the successful genomic integration to be identified by a simple restriction digest. Representative PCRs from 225 lines confirm that two lines, 7A and 17C incorporated the ssODN by homologous recombination. Animals where the wild-type genome was retained (6C, 17B, 7B & 18A) were resistant to BssHII digestion and could be used as CRISPR controls. For the two positives lines 7A and 17C, two digestion bands of 350 bp and 216 bp were obtained (Arrows, lanes 8 & 10), confirming successful genome editing. This was further confirmed by DNA sequencing of the casp genomic region. (E) Survival plot for casp^K551R transgenic lines for both uninfected and infected. The homozygous casp^K551R mutant lines show a shortened lifespan. This lifespan is further shortened when animals are infected with gram-negative E. coli. The data were analyzed using Log-rank test of trend (F) Tabulation of median lifespan for Casp SUMO mutants along with all relevant controls.Values for the Median lifespan extracted from survival data were plotted using GraphPad 8.