PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

Chemical Epigenetic Regulation of Adeno-Associated Virus Delivered Transgenes.

Adeno-associated virus (AAV) is a powerful gene therapy vector that has been used in several FDA-approved therapies as well as in multiple clinical trials. This vector has high therapeutic versatility with the ability to deliver genetic payloads to a variety of human tissue types, yet there is currently a lack of transgene expression control once the virus is administered. There are also times when transgene expression is too low for the desired therapeutic outcome, necessitating high viral dose administration resulting in possible immunological complications. Herein, we validate a chemically controllable AAV transgene expression technology in vitro that utilizes bifunctional molecules known as chemical epigenetic modifiers (CEMs). These compounds employ endogenous epigenetic machinery to specifically enhance transgene expression of episomal DNA. A recombinant AAV (rAAV) was designed to both deliver the reporter transgene as well as deliver a synthetic zinc finger (ZFs) protein fused to FK506 binding protein (FKBP). These synthetic ZFs target a DNA-binding array sequence upstream of the promoter expressing the AAV transgene to specifically enhance AAV transgene expression in the presence of a CEM. The transcriptional activating compound CEM87 functions by recruiting the epigenetic transcription activator bromodomain-containing protein 4 (BRD4), increasing AAV transgene activity up to fivefold in a dose-dependent manner in HEK293T cells. The highest levels of transgene product activity are seen 24 h following CEM87 treatment. Additionally, the CEM87-mediated enhancement of different transgene products with either Luciferase or green fluorescent protein (GFP) was observed in multiple cell lines and enhancement of transgene expression was capsid serotype independent. The impact of CEM87 activity can be disrupted through drug removal or chemical recruitment site competition with FK506, thus demonstrating the reversibility of the impact of CEM87 on transgene expression. Collectively, this chemically controllable rAAV transgene technology provides temporal gene expression control that could increase the safety and efficiency of AAV-based research and therapies.

Detection of SARS-CoV-2 in the air in Indian hospitals and houses of COVID-19 patients.

To understand the transmission characteristics of severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) through air, samples from different locations occupied by coronavirus disease (COVID-19) patients were analyzed. Three sampling strategies were used to understand the presence of virus in the air in different environmental conditions. In the first strategy, which involved hospital settings, air samples were collected from several areas of hospitals like COVID-intensive-care units (ICUs), nurse-stations, COVID-wards, corridors, non-COVID-wards, personal protective equipment (PPE) doffing areas, COVID rooms, out-patient (OP) corridors, mortuary, COVID casualty areas, non-COVID ICUs and doctors' rooms. Out of the 80 air samples collected from 6 hospitals from two Indian cities- Hyderabad and Mohali, 30 samples showed the presence of SARS-CoV-2 nucleic acids. In the second sampling strategy, that involved indoor settings, one or more COVID-19 patients were asked to spend a short duration of time in a closed room. Out of 17 samples, 5 samples, including 4 samples collected after the departure of three symptomatic patients from the room, showed the presence of SARS-CoV-2 nucleic acids. In the third strategy, involving indoor settings, air samples were collected from rooms of houses of home-quarantined COVID-19 patients and it was observed that SARS-CoV-2 RNA could be detected in the air in the rooms occupied by COVID-19 patients but not in the other rooms of the houses. Taken together, we observed that the air around COVID-19 patients frequently showed the presence of SARS-CoV-2 RNA in both hospital and indoor residential settings and the positivity rate was higher when 2 or more COVID-19 patients occupied the room. In hospitals, SARS-CoV-2 RNA could be detected in ICUs as well as in non-ICUs, suggesting that the viral shedding happened irrespective of the severity of the infection. This study provides evidence for the viability of SARS-CoV-2 and its long-range transport through the air. Thus, airborne transmission could be a major mode of transmission for SARS-CoV-2 and appropriate precautions need to be followed to prevent the spread of infection through the air.