A temperature-sensitive and interferon-silent Sendai virus vector for CRISPR-Cas9 delivery and gene editing in primary human cells.

The transformative potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and interferon-silent Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types without inducing IFN responses. ts SeV demonstrates unprecedented transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49fhigh stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help to further expand the possibilities in personalized medicine and the treatment of genetic disorders.

TRIM25 Binds RNA to Modulate Cellular Anti-viral Defense.

TRIM25 is a multi-domain, RING-type E3 ubiquitin ligase of the tripartite motif family that has important roles in multiple RNA-dependent processes. In particular, TRIM25 functions as an effector of RIG-I and ZAP, which are innate immune sensors that recognize viral RNA and induce ubiquitin-dependent anti-viral response mechanisms. TRIM25 is reported to also bind RNA, but the molecular details of this interaction or its relevance to anti-viral defense have not been elucidated. Here, we characterize the RNA-binding activity of TRIM25 and find that the protein binds both single-stranded and double-stranded RNA. Multiple regions of TRIM25 contribute to this functionality, including the C-terminal SPRY domain and a lysine-rich motif in the linker segment connecting the SPRY and coiled-coil domains. RNA binding modulates TRIM25's ubiquitination activity in vitro, its localization in cells, and its anti-viral activity. Taken together with other studies, our results indicate that RNA binding by TRIM25 has at least three important functional consequences: by enhancing ubiquitination activity, either through allosteric effects or through clustering of multiple TRIM25 molecules; by modulating the multi-domain structure of the TRIM25 dimer, and thereby structural coupling of the SPRY and RBCC elements during the ubiquitination reaction; and by facilitating subcellular localization of the E3 ligase during virus infection.