Delivering CRISPR to the HIV-1 reservoirs.

Human immunodeficiency virus type 1 (HIV-1) infection is well known as one of the most complex and difficult viral infections to cure. The difficulty in developing curative strategies arises in large part from the development of latent viral reservoirs (LVRs) within anatomical and cellular compartments of a host. The clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system shows remarkable potential for the inactivation and/or elimination of integrated proviral DNA within host cells, however, delivery of the CRISPR/Cas9 system to infected cells is still a challenge. In this review, the main factors impacting delivery, the challenges for delivery to each of the LVRs, and the current successes for delivery to each reservoir will be discussed.

Off-Target Analysis in Gene Editing and Applications for Clinical Translation of CRISPR/Cas9 in HIV-1 Therapy.

As genome-editing nucleases move toward broader clinical applications, the need to define the limits of their specificity and efficiency increases. A variety of approaches for nuclease cleavage detection have been developed, allowing a full-genome survey of the targeting landscape and the detection of a variety of repair outcomes for nuclease-induced double-strand breaks. Each approach has advantages and disadvantages relating to the means of target-site capture, target enrichment mechanism, cellular environment, false discovery, and validation of bona fide off-target cleavage sites in cells. This review examines the strengths, limitations, and origins of the different classes of off-target cleavage detection systems including anchored primer enrichment (GUIDE-seq), in situ detection (BLISS), in vitro selection libraries (CIRCLE-seq), chromatin immunoprecipitation (ChIP) (DISCOVER-Seq), translocation sequencing (LAM PCR HTGTS), and in vitro genomic DNA digestion (Digenome-seq and SITE-Seq). Emphasis is placed on the specific modifications that give rise to the enhanced performance of contemporary techniques over their predecessors and the comparative performance of techniques for different applications. The clinical relevance of these techniques is discussed in the context of assessing the safety of novel CRISPR/Cas9 HIV-1 curative strategies. With the recent success of HIV-1 and SIV-1 viral suppression in humanized mice and non-human primates, respectively, using CRISPR/Cas9, rigorous exploration of potential off-target effects is of critical importance. Such analyses would benefit from the application of the techniques discussed in this review.

Backbone and side-chain chemical shift assignments for the C-terminal domain of Tcb2, a cytoskeletal calcium-binding protein from Tetrahymena thermophila.

Tcb2 is a putative calcium-binding protein from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila. It has been hypothesized to participate in several calcium-mediated processes in Tetrahymena, including ciliary movement, cell cortex signaling, and pronuclear exchange. Sequence analysis suggests that the protein belongs to the calmodulin family, with N- and C-terminal domains connected by a central linker, and two helix-loop-helix motifs in each domain. However, its calcium-binding properties, structure and precise biological function remain unknown. Interestingly, Tcb2 is a major component of unique contractile fibers isolated from the Tetrahymena cytoskeleton; in these fibers, addition of calcium triggers an ATP-independent type of contraction. Here we report the (1)H, (13)C and (15)N backbone and side-chain chemical shift assignments of the C-terminal domain of the protein (Tcb2-C) in the absence and presence of calcium ions. (1)H-(15)N HSQC spectra show that the domain is well folded both in the absence and presence of calcium, and undergoes a dramatic conformational change upon calcium addition. Secondary structure prediction from chemical shifts reveals an architecture encountered in other calcium-binding proteins, with paired EF-hand motifs connected by a flexible linker. These studies represent a starting point for the determination of the high-resolution solution structure of Tcb2-C at both low and high calcium levels, and, together with additional structural studies on the full-length protein, will help establish the molecular basis of Tcb2 function and unique contractile properties.