A comprehensive update to the Mycobacterium tuberculosis H37Rv reference genome.

H37Rv is the most widely used Mycobacterium tuberculosis strain, and its genome is globally used as the M. tuberculosis reference sequence. Here, we present Bact-Builder, a pipeline that uses consensus building to generate complete and accurate bacterial genome sequences and apply it to three independently cultured and sequenced H37Rv aliquots of a single laboratory stock. Two of the 4,417,942 base-pair long H37Rv assemblies are 100% identical, with the third differing by a single nucleotide. Compared to the existing H37Rv reference, the new sequence contains ~6.4 kb additional base pairs, encoding ten new regions that include insertions in PE/PPE genes and new paralogs of esxN and esxJ, which are differentially expressed compared to the reference genes. New sequencing and de novo assemblies with Bact-Builder confirm that all 10 regions, plus small additional polymorphisms, are also present in the commonly used H37Rv strains NR123, TMC102, and H37Rv1998. Thus, Bact-Builder shows promise as an improved method to perform accurate and reproducible de novo assemblies of bacterial genomes, and our work provides important updates to the primary M. tuberculosis reference genome.

Decontaminating N95 respirators during the Covid-19 pandemic: simple and practical approaches to increase decontamination capacity, speed, safety and ease of use.

BACKGROUND: The COVID-19 pandemic has caused a severe shortage of personal protective equipment (PPE), especially N95 respirators. Efficient, effective and economically feasible methods for large-scale PPE decontamination are urgently needed. AIMS: (1) to develop protocols for effectively decontaminating PPE using vaporized hydrogen peroxide (VHP); (2) to develop novel approaches that decrease set up and take down time while also increasing decontamination capacity (3) to test decontamination efficiency for N95 respirators heavily contaminated by makeup or moisturizers. METHODS: We converted a decommissioned Biosafety Level 3 laboratory into a facility that could be used to decontaminate N95 respirators. N95 respirators were hung on metal racks, stacked in piles, placed in paper bags or covered with makeup or moisturizer. A VHP VICTORYTM unit from STERIS was used to inject VHP into the facility. Biological and chemical indicators were used to validate the decontamination process. FINDINGS: N95 respirators individually hung on metal racks were successfully decontaminated using VHP. N95 respirators were also successfully decontaminated when placed in closed paper bags or if stacked in piles of up to six. Stacking reduced the time needed to arrange N95 respirators for decontamination by approximately two-thirds while almost tripling facility capacity. Makeup and moisturizer creams did not interfere with the decontamination process. CONCLUSIONS: Respirator stacking can reduce the hands-on time and increase decontamination capacity. When personalization is needed, respirators can be decontaminated in labeled paper bags. Make up or moisturizers do not appear to interfere with VHP decontamination.

Target Discovery for Precision Medicine Using High-Throughput Genome Engineering.

Over the past few years, programmable RNA-guided nucleases such as the CRISPR/Cas9 system have ushered in a new era of precision genome editing in diverse model systems and in human cells. Functional screens using large libraries of RNA guides can interrogate a large hypothesis space to pinpoint particular genes and genetic elements involved in fundamental biological processes and disease-relevant phenotypes. Here, we review recent high-throughput CRISPR screens (e.g. loss-of-function, gain-of-function, and targeting noncoding elements) and highlight their potential for uncovering novel therapeutic targets, such as those involved in cancer resistance to small molecular drugs and immunotherapies, tumor evolution, infectious disease, inborn genetic disorders, and other therapeutic challenges.

Application of sensitive and specific molecular methods to uncover global dissemination of the major RDRio Sublineage of the Latin American-Mediterranean Mycobacterium tuberculosis spoligotype family.

The Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosis is believed to be the cause of approximately 15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RD(Rio). Using the Brazilian strains, we pinpoint an Ag85C(103) single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism [RFLP] analysis) that correctly identified all LAM family strains. Importantly, all RD(Rio) strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between "wild-type" and RD(Rio) strains, were then used to analyze an international collection of M. tuberculosis strains. RD(Rio) M. tuberculosis was identified from four continents involving 11 countries. Phylogenetic analysis of the IS6110-RFLP patterns from representative RD(Rio) and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C(103) SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS6110 element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RD(Rio) strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RD(Rio) sublineage.