CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs

Abstract

Current antiretroviral therapy does not eliminate the integrated and transcriptionally silent HIV-1 provirus in latently infected cells. Recently, a “shock and kill” strategy has been extensively explored to eradicate the HIV-1 latent reservoirs for a permanent cure of AIDS. The therapeutic efficacy of currently used agents remains disappointing because of low efficiency, non-specificity and cellular toxicity. Here we present a novel catalytically-deficient Cas9-synergistic activation mediator (dCas9-SAM) technology to selectively, potently and persistently reactivate the HIV-1 latent reservoirs. By screening 16 MS2-mediated single guide RNAs, we identified long terminal repeat (LTR)-L and O that surround the enhancer region (-165/-145 for L and -92/-112 for O) and induce robust reactivation of HIV-1 provirus in HIV-1 latent TZM-bI epithelial, Jurkat T lymphocytic and CHME5 microglial cells. This compulsory reactivation induced cellular suicide via toxic buildup of viral proteins within HIV-1 latent Jurkat T and CHME5 microglial cells. These results suggest that this highly effective and target-specific dCas9-SAM system can serve as a novel HIV-latency-reversing therapeutic tool for the permanent elimination of HIV-1 latent reservoirs.

The Chromatin Remodeling ComponentArid1aIs a Suppressor of Spontaneous Mammary Tumors in Mice

“…Significant differences were calculated using a twotailed Student's t-test (** P , 0. Gilbert et al 2014;Chavez et al 2015;Konermann et al 2015;Zhang et al 2015).…”

“…Sporadic breast cancer (i.e., not associated with inherited neoplasia-driving mutations) accounts for the vast majority of breast cancer cases in the U.S. (80-85%) (American Cancer Society 2014). Although ARID1A has not yet been widely recognized as a key suppressor of breast carcinogenesis, it is heterozygously deleted in a substantial fraction of tumors (Cornen et al 2012;Mamo et al 2012), and low ARID1A expression in tumors of patients with breast cancer correlates significantly with poorer prognosis and overall survival (Mamo et al 2011;Zhao et al 2014;Cho et al 2015;Zhang et al 2015). Here, we report functional evidence that Arid1a loss is critical for mammary tumorigenesis in a mouse model of spontaneous breast cancer and present data on how this leads to deregulated cancer cell growth.…”

CRISPRs encounter Cocktail: a dawn for curing HIV/AIDS

“…The combination of Cas9 activators and latency-reversing compounds could promote latent HIV-1 gene activation, which has been proved by dCas9-VP64 and a superior dCas9-SAM systems [27][28][29]. Compare to chemical activating agents, CRISPR/Cas9 system is safer and more efficient though, the balance of "shock" and "kill" is difficult to master.…”

Gene Editing for Treatment of Neurological Infections

“…Owing to their poor efficiency and tendencies towards nonspecific and toxic effects, the therapeutic efficacy of the agents currently used in this approach is disappointing. Zhang et al [44] reported a novel catalytically deficient Cas9-synergistic activation mediator technology to allow more selective and potent reactivation of latent HIV-1. Multiple gRNAs were screened and HIV-1 regions were identified that permit induction of robust reactivation of HIV-1 provirus in latently infected cell lines, including the TZM-bI epithelial HeLa cell derivative, Jurkat T lymphocytes, and CHME5 microglia [44].…”