A CRISPR/Cas9 assisted strategy for the conditional expression of human NF-kappaB c-Rel cDNA in mouse T cells: design, prospects, and challenges.

Nuclear factor-κB protein c-Rel is a critical regulator of autoimmune diabetes. We found that c-Rel O-GlcNAcylation at serine-350 increases with hyperglycemia, which results in increased transcription of proautoimmune Th1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-γ), and decreased transcription of the T regulatory cell transcription factor forkhead box 3 (FOXP3). To further study the translational relevance of c-Rel S350 O-GlcNAcylation in autoimmune diabetes, we sought to generate transgenic non-obese diabetic (NOD) mice conditionally expressing wildtype or mutant S350A human c-Rel cDNA in T cells downstream of the endogenous mouse REL promoter. We used CRISPR-Cas9 gene editing to insert a unique designer cassette containing floxed mouse c-Rel cDNA-STOP sequence to maintain whole body c-Rel expression, followed by a linker and human c-Rel cDNA-STOP sequence. Using comprehensive PCR analyses and high-throughput sequencing, we confirmed successful insertion of the cassette at the mouse REL locus and the expected deletion of the mouse c-Rel cDNA specifically in T cells following CD4-Cre mating. Additional characterization revealed that the knock-in transgenic mice lacked endogenous mouse c-Rel, further confirming desired interference with its natural start codon. Unexpectedly, these mice lacked mouse and human c-Rel protein expression from inserted cDNAs, which mechanistically correlated with increased CpG methylation of the c-Rel promoter region. Thus, our study presents a unique, universal molecular design and method for the generation of conditional knock-in transgenic mice expressing human genes at the endogenous mouse promoter. It also reveals a potential locus-specific challenge that may arise during the development of such novel transgenic mouse models.