CRISPR/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli.

Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.

Predicting Drug Resistance Using Deep Mutational Scanning.

Drug resistance is a major healthcare challenge, resulting in a continuous need to develop new inhibitors. The development of these inhibitors requires an understanding of the mechanisms of resistance for a critical mass of occurrences. Recent genome editing technologies based on high-throughput DNA synthesis and sequencing may help to predict mutations resulting in resistance by testing large mutagenesis libraries. Here we describe the rationale of this approach, with examples and relevance to drug development and resistance in malaria.

Unveiling Cellular Identities and Gene Expression Pathways in Overlapping Myocarditis and Myositis.

Immune checkpoint therapies can drive antitumor responses and benefit patients but can also induce life-threatening immune-related adverse events such as myocarditis and myositis. These immune-related adverse events are rare but carry substantial morbidity and mortality. In this issue, Siddiqui and colleagues use single-cell RNA and T-cell receptor sequencing to identify novel cellular subsets and propose various mechanisms that could contribute to the pathogenesis of immune checkpoint inhibitor-associated myocarditis and myositis. These new insights should help move the field toward the development of improved treatment and prevention options, ultimately improving patient outcomes. See related article by Siddiqui et al., p. 964 (1).

Genetic analysis of multiple primary melanomas arising within the boundaries of congenital nevi depigmentosa.

Here, we present a rare case of a patient who developed multiple primary melanomas within the boundaries of two nevi depigmentosa. The melanomas were excised, and as a preventive measure, the remainder of the nevi depigmentosa were removed. We performed whole-exome sequencing on excised tissue from the nevus depigmentosus, adjacent normal skin, and saliva to explain this intriguing phenomenon. We also performed a GeneTrails Comprehensive Solid Tumor Panel analysis on one of the melanoma tissues. Genetic analysis revealed germline MC1R V92M and TYR R402Q polymorphisms and a MET E168D germline mutation that may have increased the risk of melanoma development. This genetic predisposition, combined with a patient-reported history of substantial sun exposure and sunburns, which were more severe within the boundaries of the nevi depigmentosa due to the lack of photoprotective melanin, produced numerous somatic mutations in the melanocytes of the nevi depigmentosa. Fitting with this paradigm for melanoma development in chronically sun-damaged skin, the patient's melanomas harbored somatic mutations in CDKN2A (splice site), NF1, and ATRX and had a tumor mutation burden in the 90-95th percentile for melanoma.

Determinants for Efficient Editing with Cas9-Mediated Recombineering in Escherichia coli.

In E. coli, editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency. These effects on editing efficiency were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs, as well as possible differences in binding of Cas9:gRNA complexes to their target sites, account for the observed variations in editing efficiency between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.

Genomic Deoxyxylulose Phosphate Reductoisomerase (DXR) Mutations Conferring Resistance to the Antimalarial Drug Fosmidomycin in E. coli.

Sequence to activity mapping technologies are rapidly developing, enabling the generation and isolation of mutations conferring novel phenotypes. Here we used the CRISPR enabled trackable genome engineering (CREATE) technology to investigate the inhibition of the essential ispC gene in its native genomic context in Escherichia coli. We created a full saturation library of 33 sites proximal to the ligand binding pocket and challenged this library with the antimalarial drug fosmidomycin, which targets the ispC gene product, DXR. This selection is especially challenging since it is relatively weak in E. coli, with multiple naturally occurring pathways for resistance. We identified several previously unreported mutations that confer fosmidomycin resistance, in highly conserved sites that also exist in pathogens including the malaria-inducing Plasmodium falciparum. This approach may have implications for the isolation of resistance-conferring mutations and may affect the design of future generations of fosmidomycin-based drugs.