Genome-wide gene expression tuning reveals diverse vulnerabilities of M. tuberculosis Graphical abstract Highlights d Titratable CRISPRi enables quantification of target vulnerability in mycobacteria d Essential genes and processes vary widely in their vulnerability d Differential vulnerability predicts differential antibacterial susceptibility d Generalizable approach allows prioritization of high-value targets for drug discovery

Mycobacterium tuberculosis (Mtb) infection is notoriously difficult to treat. Treatment efficacy is limited by Mtb’s intrinsic drug resistance, as well as its ability to evolve acquired resistance to all antituberculars in clinical use. A deeper understanding of the bacterial pathways that influence drug efficacy could facilitate the development of more effective therapies, identify new mechanisms of acquired resistance, and reveal overlooked therapeutic opportunities. Here we developed a CRISPR interference chemical-genetics platform to titrate the expression of Mtb genes and quantify bacterial fitness in the presence of different drugs. We discovered diverse mechanisms of intrinsic drug resistance, unveiling hundreds of potential targets for synergistic drug combinations. Combining chemical genetics with comparative genomics of Mtb clinical isolates, we further identified several previously unknown mechanisms of acquired drug resistance, one of which is associated with a multidrug-resistant tuberculosis outbreak in South America. Lastly, we found that the intrinsic resistance factor whiB7 was inactivated in an entire Mtb sublineage endemic to Southeast Asia, presenting an opportunity to potentially repurpose the macrolide antibiotic clarithromycin to treat tuberculosis. This chemical-genetic map provides a rich resource to understand drug efficacy in Mtb and guide future tuberculosis drug development and treatment.

The advent of next generation sequencing (NGS) in 2010 has transformed medicine and particularly the growing field of monogenic inborn errors of immunity, including primary immunodeficiencies (PID). NGS has facilitated the discovery of novel disease-causing genes and the genetic diagnosis of patients with PID. Whole-exome sequencing (WES) is presently the most cost-effective approach for PID research and diagnostics, though whole genome sequencing (WGS) offers several advantages. The scientific or diagnostic challenge consists in selecting one or two candidate variants among thousands of NGS calls. Variant- and gene-level computational methods as well as immunological hypotheses can help to narrow down this genome-wide search. The key to success is a well-informed genetic hypothesis on three key aspects: mode of inheritance, clinical penetrance, and genetic heterogeneity of the condition. This determines the search strategy and the frequency cut-offs for candidate alleles. Subsequent functional validation of the disease-causing effect of the candidate variant is critical. Even the most up-to-date dry lab cannot clinch this validation without a seasoned wet lab. The multifariousness of variations entails an experimental rigor even greater than traditional Sanger sequencing-based approaches, in order not to assign PID to false positives. Finding the needle in the haystack takes patience, prudence, and discernment.

Deficiency of adenosine deaminase 2 (DADA2) is caused by biallelic deleterious mutations in DADA2 results in variable autoinflammation and vasculopathy (recurrent fevers, livedo reticularis, polyarteritis nodosa, lacunar ischemic strokes, and intracranial hemorrhages), immunodeficiency and bone marrow failure. Tumor necrosis factor-α blockade is the treatment of choice for the autoinflammation and vascular manifestations. Hematopoietic stem cell transplantation (HSCT) represents a potential definitive treatment. We present a cohort of 14 patients from 6 countries who received HSCT for DADA2. Indication for HSCT was bone marrow dysfunction or immunodeficiency. Six of 14 patients had vasculitis pre-HSCT. The median age at HSCT was 7.5 years. Conditioning regimens were myeloablative (9) and reduced intensity (5). Donors were HLA-matched sibling (n = 1), HLA-matched unrelated (n = 9), HLA-mismatched unrelated (n = 3), and HLA haploidentical sibling (n = 1). All patients are alive and well with no new vascular events and resolution of hematological and immunological phenotype at a median follow-up of 18 months (range, 5 months to 13 years). Plasma ADA2 enzyme activity normalized in those tested post-HSCT (7/7), as early as day +14 (myeloid engraftment). Post-HSCT hematological autoimmunity (cytopenias) was reported in 4 patients, acute graft-versus-host disease grade 1 in 2, grade 2 in 3, and grade 3-4 in 1, and moderate chronic graft-versus-host disease in 1 patient. In conclusion, in 14 patients, HSCT was an effective and definitive treatment of DADA2.