HIV-1 cure strategies: why CRISPR?

INTRODUCTION: Antiretroviral therapy (ART) has transformed prognoses for HIV-1-infected individuals but requires lifelong adherence to prevent viral resurgence. Targeted elimination or permanent deactivation of the latently infected reservoir harboring integrated proviral DNA, which drives viral rebound, is a major focus of HIV-1 research. AREAS COVERED: This review covers the current approaches to developing curative strategies for HIV-1 that target the latent reservoir. Discussed herein are shock and kill, broadly neutralizing antibodies (bNAbs), block and lock, Chimeric antigen receptor (CAR) T cells, immune checkpoint modulation, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) coreceptor ablation, and CRISPR/Cas9 proviral excision. Emphasis is placed on CRISPR/Cas9 proviral excision/inactivation. Recent advances and future directions toward discovery and translation of HIV-1 therapeutics are discussed. EXPERT OPINION: CRISPR/Cas9 proviral targeting fills a niche amongst HIV-1 cure strategies by directly targeting the integrated provirus without the necessity of an innate or adaptive immune response. Each strategy discussed in this review has shown promising results with the potential to yield curative or adjuvant therapies. CRISPR/Cas9 is singular among these in that it addresses the root of the problem, integrated proviral DNA, with the capacity to permanently remove or deactivate the source of HIV-1 recrudescence.

Designing Safer CRISPR/Cas9 Therapeutics for HIV: Defining Factors That Regulate and Technologies Used to Detect Off-Target Editing.

Human immunodeficiency virus type-1 (HIV-1) infection has resulted in the death of upward of 39 million people since being discovered in the early 1980s. A cure strategy for HIV-1 has eluded scientists, but gene editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) offer a new approach to developing a cure for HIV infection. While the CRISPR/Cas9 system has been used successfully in a number of different types of studies, there remains a concern for off-target effects. This review details the different aspects of the Cas9 system and how they play a role in off-target events. In addition, this review describes the current technologies available for detecting off-target cleavage events and their advantages and disadvantages. While some studies have utilized whole genome sequencing (WGS), this method sacrifices depth of coverage for interrogating the whole genome. A number of different approaches have now been developed to take advantage of next generation sequencing (NGS) without sacrificing depth of coverage. This review highlights four widely used methods for detecting off-target events: (1) genome-wide unbiased identification of double-stranded break events enabled by sequencing (GUIDE-Seq), (2) discovery of in situ Cas off-targets and verification by sequencing (DISCOVER-Seq), (3) circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-Seq), and (4) breaks labeling in situ and sequencing (BLISS). Each of these technologies has advantages and disadvantages, but all center around capturing double-stranded break (DSB) events catalyzed by the Cas9 endonuclease. Being able to define off-target events is crucial for a gene therapy cure strategy for HIV-1.

Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites.

Viral latency of human immunodeficiency virus type 1 (HIV-1) has become a major hurdle to a cure in the highly effective antiretroviral therapy (ART) era. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has successfully been demonstrated to excise or inactivate integrated HIV-1 provirus from infected cells by targeting the long terminal repeat (LTR) region. However, the guide RNAs (gRNAs) have classically avoided transcription factor binding sites (TFBSs) that are readily observed and known to be important in human promoters. Although conventionally thought unfavorable due to potential impact on human promoters, our computational pipeline identified gRNA sequences that were predicted to inactivate HIV-1 transcription by targeting the nuclear factor κB (NF-κB) binding sites (gNFKB0, gNFKB1) with a high safety profile (lack of predicted or observed human edits) and broad-spectrum activity (predicted coverage of known viral sequences). Genome-wide, unbiased identification of double strand breaks (DSBs) enabled by sequencing (GUIDE-seq) showed that the gRNAs targeting NF-κB binding sites had no detectable CRISPR-induced off-target edits in HeLa cells. 5' LTR-driven HIV-1 transcription was significantly reduced in three HIV-1 reporter cell lines. These results demonstrate a working model to specifically target well-known TFBSs in the HIV-1 LTR that are readily observed in human promoters to reduce HIV-1 transcription with a high-level safety profile and broad-spectrum activity.

The Influence of the Electronic Structure Method on Intersystem Crossing Dynamics. The Case of Thioformaldehyde.

The ability of different electronic structure methods to correctly describe intersystem crossing dynamics is evaluated, using thioformaldehyde as a test case. Mischievously, all methods considered-ranging from the multireference methods MRCISD, MS-CASPT2, or SA-CASSCF, to the single-reference methods ADC(2), CC2, and TDDFT in different flavors-provide the same state ordering and energies of the low-lying singlet and triplet electronic excited states within an acceptable error of 0.2-0.3 eV. However, the outcome of the nonadiabatic simulations after excitation to the lowest S1 (1 nπ*) state are dramatically different. While MS-CASPT2, ADC(2), BP86, and PBE do not transfer population to the triplet states within 500 fs-consistent with experimental evidence-SA-CASSCF, B3LYP, and BHHLYP predict intersystem crossing yields between 3% and 21% within the same time. The different excited state dynamics can be rationalized by inspecting potential energy profiles along the C-S bond stretch mode and single-triplet energy gaps. It is found that already at a C-S bond length of 1.9 Å, all the single-reference methods struggle to describe the correct asymptotic behavior of the potentials. Moreover, some methods, including SA-CASSCF, obtain incorrect 1 nπ*-3 ππ* energy gaps, leading to compensation of errors (ADC(2), BP86, PBE), or wrong dynamics (SA-CASSCF, B3LYP, BHHLYP). Only the accurate MRCISD and MS-CASPT2 methods are able to describe the C-S bond correctly and thus able to deliver the correct potential energy surfaces and dynamics for the right reason. A correlation with the amount of Hartree-Fock exchange in the density functional and the ease to access the 3 ππ* state from the 1 nπ* are able to explain the different behavior observed for GGA and hybrid functionals. It is thus illustrated that even in the case of a simple molecule, like CH2S, the sole assessment of vertical excitation energies as reliability predictors for nonadiabatic dynamics is inadequate. The reason is that ISC does not occur at the FC geometry, but rather at distorted geometries where the singlet-triplet gaps become small. Hence, a characterization of the potential energy surfaces beyond the Franck-Condon region is mandatory.

Assessing Excited State Energy Gaps with Time-Dependent Density Functional Theory on Ru(II) Complexes.

A set of density functionals coming from different rungs on Jacob's ladder is employed to evaluate the electronic excited states of three Ru(II) complexes. While most studies on the performance of density functionals compare the vertical excitation energies, in this work we focus on the energy gaps between the electronic excited states, of the same and different multiplicity. Excited state energy gaps are important for example to determine radiationless transition probabilities. Besides energies, a functional should deliver the correct state character and state ordering. Therefore, wave function overlaps are introduced to systematically evaluate the effect of different functionals on the character of the excited states. As a reference, the energies and state characters from multistate second-order perturbation theory complete active space (MS-CASPT2) are used. In comparison to MS-CASPT2, it is found that while hybrid functionals provide better vertical excitation energies, pure functionals typically give more accurate excited state energy gaps. Pure functionals are also found to reproduce the state character and ordering in closer agreement to MS-CASPT2 than the hybrid functionals.

Trajectory Surface-Hopping Dynamics Including Intersystem Crossing in [Ru(bpy)3]2.

Surface-hopping dynamics coupled to linear response TDDFT and explicit nonadiabatic and spin-orbit couplings have been used to model the ultrafast intersystem crossing (ISC) dynamics in [Ru(bpy)3]2+. Simulations using an ensemble of trajectories starting from the singlet metal-to-ligand charge transfer (1MLCT) band show that the manifold of 3MLCT triplet states is first populated from high-lying singlet states within 26 ± 3 fs. ISC competes with an intricate internal conversion relaxation process within the singlet manifold to the lowest singlet state. Normal-mode analysis and principal component analysis, combined with further dynamical simulations where the nuclei are frozen, unequivocally demonstrate that it is not only the high density of states and the large spin-orbit couplings of the system that promote ISC. Instead, geometrical relaxation involving the nitrogen atoms is required to allow for state mixing and efficient triplet population transfer.

Structural snapshots of the SCR reaction mechanism on Cu-SSZ-13.

The structure of copper sites in Cu-SSZ-13 during NH3-SCR was unravelled by a combination of novel operando X-ray spectroscopic techniques. Strong adsorption of NH3 on Cu, its reaction with weakly adsorbed NO from the gas phase, and slow re-oxidation of Cu(I) were proven. Thereby the SCR reaction mechanism is significantly different to that observed for Fe-ZSM-5.

High-resolution X-ray absorption spectroscopy of iron carbonyl complexes.

We apply high-energy-resolution fluorescence-detected (HERFD) X-ray absorption near-edge spectroscopy (XANES) to study iron carbonyl complexes. Mono-, bi-, and tri-nuclear carbonyl complexes and pure carbonyl complexes as well as carbonyl complexes containing hydrocarbon ligands are considered. The HERFD-XANES spectra reveal multiple pre-edge peaks with individual signatures for each complex, which could not be detected previously with conventional XANES spectroscopy. These peaks are assigned and analysed with the help of TD-DFT calculations. We demonstrate that the pre-edge peaks can be used to distinguish the different types of iron-iron interactions in carbonyl complexes. This opens up new possibilities for applying HERFD-XANES spectroscopy to probe the electronic structure of iron catalysts.

Selective catalytic reduction of NO over Fe-ZSM-5: mechanistic insights by operando HERFD-XANES and valence-to-core X-ray emission spectroscopy.

An in-depth understanding of the active site requires advanced operando techniques and the preparation of defined catalysts. We elucidate here the mechanism of the selective catalytic reduction of NO by NH3 (NH3-SCR) over a Fe-ZSM-5 zeolite catalyst. 1.3 wt % Fe-ZSM-5 with low nuclearity Fe sites was synthesized, tested in the SCR reaction and characterized by UV-vis, X-ray absorption near edge structure (XANES), and extended X-ray absorption fine structure (EXAFS) spectroscopy. Next, this defined Fe-zeolite catalyst was studied by complementary high-energy-resolution fluorescence-detected XANES (HERFD-XANES) and valence-to-core X-ray emission spectroscopy (V2C XES) under different model in situ and realistic working (operando) conditions identical to the catalyst test bench including the presence of water vapor. HERFD-XANES uncovered that the coordination (between 4 and 5), geometry (tetrahedral, partly 5-fold), and oxidation state of the Fe centers (reduced in NH3, partly in SCR mixture, slight reduction in NO) strongly changed. V2C XES supported by DFT calculations provided important insight into the chemical nature of the species adsorbed on Fe sites. The unique combination of techniques applied under realistic reaction conditions and the corresponding catalytic data unraveled the adsorption of ammonia via oxygen on the iron site. The derived reaction model supports a mechanism where adsorbed NOx reacts with ammonia coordinated to the Fe(3+) site yielding Fe(2+) whose reoxidation is slow.

The chemical sensitivity of X-ray spectroscopy: high energy resolution XANES versus X-ray emission spectroscopy of substituted ferrocenes.

X-ray spectroscopy at the metal K-edge is an important tool for understanding catalytic processes and provides insight into the geometric and electronic structures of transition metal complexes. In particular, X-ray emission-based methods such as high-energy resolution fluorescence detection (HERFD), X-ray absorption near-edge spectroscopy (XANES) and valence-to-core X-ray emission spectroscopy (V2C-XES) hold the promise of providing increased chemical sensitivity compared to conventional X-ray absorption spectroscopy. Here, we explore the ability of HERFD-XANES and V2C-XES spectroscopy to distinguish substitutions beyond the directly coordinated atoms for the example of ferrocene and selected ferrocene derivatives. The experimental spectra are assigned and interpreted through the use of density functional theory (DFT) calculations. We find that while the pre-edge peaks in the HERFD-XANES spectra are affected by substituents at the cyclopentadienyl ring containing π-bonds [A. J. Atkins, Ch. R. Jacob and M. Bauer, Chem.-Eur. J., 2012, 18, 7021], the V2C-XES spectra are virtually unchanged. The pre-edge in HERFD-XANES probes the weak transition to unoccupied metal d-orbitals, while the V2C-XES spectra are determined by dipole-allowed transitions from occupied ligand orbitals to the 1s core hole. The latter turn out to be less sensitive to changes beyond the first coordination shell.

Origin-independent calculation of quadrupole intensities in X-ray spectroscopy.

For electronic excitations in the ultraviolet and visible range of the electromagnetic spectrum, the intensities are usually calculated within the dipole approximation, which assumes that the oscillating electric field is constant over the length scale of the transition. For the short wavelengths used in hard X-ray spectroscopy, the dipole approximation may not be adequate. In particular, for metal K-edge X-ray absorption spectroscopy (XAS), it becomes necessary to include higher-order contributions. In quantum-chemical approaches to X-ray spectroscopy, these so-called quadrupole intensities have so far been calculated by including contributions depending on the square of the electric-quadrupole and magnetic-dipole transition moments. However, the resulting quadrupole intensities depend on the choice of the origin of the coordinate system. Here, we show that for obtaining an origin-independent theory, one has to include all contributions that are of the same order in the wave vector consistently. This leads to two additional contributions depending on products of the electric-dipole and electric-octupole and of the electric-dipole and magnetic-quadrupole transition moments, respectively. We have implemented such an origin-independent calculation of quadrupole intensities in XAS within time-dependent density-functional theory, and demonstrate its usefulness for the calculation of metal and ligand K-edge XAS spectra of transition metal complexes.

Ni(III) vs. Ni(II)-thiyl radical: charge-delocalisation in a binuclear Ni(III)Ni(II)-dithiolate complex.

Multi-frequency EPR spectroscopy on 61Ni-labelled samples of [Ni2(L)]3+ confirms extensive charge-delocalisation between the Ni(III) centre and thiolate donors in the Ni(II)Ni(III) complex.