Harnessing the RNA-Cleaving Properties of CRISPR-Cas13a for Applications to HIV Detection and Latency

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PROJECT SUMMARY/ABSTRACT Although much progress has been made in treating human immunodeficiency virus, the presence of a persistent population of CD4+ T cells with integrated HIV proviruses, the latent reservoir, remains the major barrier to a cure. Improved tools to directly detect and target viral RNAs could greatly support efforts to understand, quantify, and eliminate the latent reservoir. Recently, CRISPR-Cas13a (formerly referred to as C2c2), was discovered to bind single-stranded target RNAs in a sequence-specific manner and exert general RNase activity upon activation by the target RNA. This non-specific or collateral cleavage can be exploited for fluorescence-based detection of specific RNAs and has been previously used for detection of Zika and Dengue RNA viruses. However, these detection strategies required reverse transcription, amplification, and T7 transcription steps which may diminish the reproducibility of the assay and introduce biases. The current gold standard of HIV-1 RNA detection, RT-PCR, also currently requires a reverse transcription step. There is a critical need for developing new methods to directly and sensitively sense HIV RNAs both in the clinic and laboratory setting. Additionally, directly studying HIV RNAs in vivo has been challenging due to limited methods to manipulate RNAs within cells. One in vivo RNA of special interest in understanding the mechanisms of HIV latency are short, abortive TAR transcripts. They are also thought to play a role in preventing apoptosis of infected cells. Although factors that TAR RNA interacts with to modulate HIV transcription have been previously knocked-down using shRNAs, few studies have targeted nascent TAR RNA itself in the context of HIV latency and infection. The development of CRISPR-Cas13a as a molecular tool can allow us to directly detect HIV RNAs in vitro and target specific HIV RNAs in vivo. We hypothesize that rigorous optimization of CRISPR-Cas13a components (Cas13a homolog, crRNA design, and fluorescent reporter RNA) can allow for direct detection and quantification of HIV RNAs, and that CRISPR-Cas13a can be utilized in vivo to cleave short TAR RNA transcripts that may contribute to HIV latency and apoptosis. We aim to develop CRISPR-Cas13 as a versatile tool for the study and detection of HIV. Together, the proposed experiments will harness a novel CRISPR technology towards direct HIV-1 RNA detection and will elucidate RNA-based mechanisms of latency, which could help identify potential HIV cure targets.