Delineating the functions of RIPK1 in vivo through CRISPR/Cas9-mediated gene editing in mice

Abstract

Abstract RIPK1 is originally identified as a protein associated with Fas and TNFR1, which can trigger apoptosis. However, initial studies indicated that RIPK1 is dispensable for apoptotic signaling. Instead, cells lacking RIPK1 are defective in TNFR1-induced activation of NF-kB, a critical player involved in pro-survival signaling. Recent studies indicate that a certain form of necrosis-like death, necroptosis, requires RIPK1. In contrast, the Fas-associated death domain (FADD) protein appears to play an obligatory role in apoptosis induced by Fas and TNFR1. Fadd−/− mice die in utero due to RIPK1 mediated necroptosis. More recently, studies including ours demonstrated that only when both FADD and RIPK3 were deleted will Ripk1−/− mice survive to adulthood. This result indicates perinatal lethality in Ripk1−/− mice is due to not only FADD-mediated apoptosis but also RIPK3-dependent necrosis. Interestingly, we found that in adult T cells RIPK1 only suppresses apoptosis. To facilitate the dissection of the multiple functions of RIPK1, we performed in vivo gene editing using the CRISPR/Cas9 technology. This allows selective interruption of one functional domain/motif of RIPK1. Using these novel mouse models, our data reveals a previously unappreciated function for RIPK1 in promoting apoptosis.