STING is essential for control of infections and for tumor immunosurveillance, but can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER), and traffics following stimulation to ERGIC/Golgi where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PI3P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP leading to increased ER PI3P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.
The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences.
Type 3 Pol III promoters such as U6 are widely used for expression of small RNAs, including short hairpin RNA for RNAi applications and guide RNA in CRISPR genome-editing platforms. RNA polymerase III uses a T-stretch as termination signal, but the exact properties have not been thoroughly investigated. Here, we systematically measured the in vivo termination efficiency and the actual site of termination for different T-stretch signals in three commonly used human Pol III promoters (U6, 7SK, and H1). Both the termination efficiency and the actual termination site depend on the T-stretch signal. The T4 signal acts as minimal terminator, but full termination efficiency is reached only with a T-stretch of ≥6. The termination site within the T-stretch is quite heterogeneous, and consequently small RNAs have a variable U-tail of 1–6 nucleotides. We further report that such variable U-tails can have a significant negative effect on the functionality of the crRNA effector of the CRISPR-AsCpf1 system. We next improved these crRNAs by insertion of the HDV ribozyme to avoid U-tails. This study provides detailed design guidelines for small RNA expression cassettes based on Pol III.
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