Lineage tracing and analog recording in mammalian cells by single-site DNA writing.

Studying cellular and developmental processes in complex multicellular organisms can require the non-destructive observation of thousands to billions of cells deep within an animal. DNA recorders address the staggering difficulty of this task by converting transient cellular experiences into mutations at defined genomic sites that can be sequenced later in high throughput. However, existing recorders act primarily by erasing DNA. This is problematic because, in the limit of progressive erasure, no record remains. We present a DNA recorder called CHYRON (Cell History Recording by Ordered Insertion) that acts primarily by writing new DNA through the repeated insertion of random nucleotides at a single locus in temporal order. To achieve in vivo DNA writing, CHYRON combines Cas9, a homing guide RNA and the template-independent DNA polymerase terminal deoxynucleotidyl transferase. We successfully applied CHYRON as an evolving lineage tracer and as a recorder of user-selected cellular stimuli.

Dystrophinopathy muscle biopsies in the genetic testing ERA: One center's data.

INTRODUCTION: Comprehensive genetic testing for dystrophinopathy can detect ∼95% of pathogenic variants in the dystrophin gene (DMD) and is often the preferred diagnostic approach. METHODS: We reviewed pathology reports for muscle biopsies evaluated at the University of Iowa with a pathological diagnosis of dystrophinopathy based on dystrophic histopathology and abnormal immunofluorescence staining: reduced to absent dystrophin, expression of utrophin, and loss of neuronal nitric oxide synthase. RESULTS: The percentage of muscle biopsies with dystrophinopathy has been stable since 1997. Among 2,298 biopsies evaluated between 2011 and 2016, 72 (3.1%) had pathologic features of dystrophinopathy. Median age at biopsy was 8 years (range, 0.66-84). Half had undergone DMD genetic testing prior to biopsy. Clinical phenotypes recorded on requisitions were typical of muscular dystrophy for 57 (79%) biopsies. DISCUSSION: Muscle biopsy continues to play an important role in the diagnosis of dystrophinopathy, particularly in patients with later symptom onset, comorbidities, or normal DMD genetic testing results. Muscle Nerve, 2018.

Pervasiveness of UVC254-resistant Geobacillus strains in extreme environments.

We have characterized a broad collection of extremophilic bacterial isolates from a deep subsurface mine, compost dumping sites, and several hot spring ecosystems. Spore-forming strains isolated from these environments comprised both obligate thermophiles/thermotolerant species (growing at > 55 °C; 240 strains) and mesophiles (growing at 15 to 40 °C; 12 strains). An overwhelming abundance of Geobacillus (81.3%) and Bacillus (18.3%) species was observed among the tested isolates. 16S rRNA sequence analysis documented the presence of 24 species among these isolates, but the 16S rRNA gene was shown to possess insufficient resolution to reliably discern Geobacillus phylogeny. gyrB-based phylogenetic analyses of nine strains revealed the presence of six known Geobacillus and one novel species. Multilocus sequence typing analyses based on seven different housekeeping genes deduced from whole genome sequencing of nine strains revealed the presence of three novel Geobacillus species. The vegetative cells of 41 Geobacillus strains were exposed to UVC254, and most (34 strains) survived 120 J/m2, while seven strains survived 300 J/m2, and cells of only one Geobacillus strain isolated from a compost facility survived 600 J/m2. Additionally, the UVC254 inactivation kinetics of spores from four Geobacillus strains isolated from three distinct geographical regions were evaluated and compared to that of a spacecraft assembly facility (SAF) clean room Geobacillus strain. The purified spores of the thermophilic SAF strain exhibited resistance to 2000 J/m2, whereas spores of two environmental Geobacillus strains showed resistance to 1000 J/m2. This study is the first to investigate UV resistance of environmental, obligately thermophilic Geobacillus strains, and also lays the foundation for advanced understanding of necessary sterilization protocols practiced in food, medical, pharmaceutical, and aerospace industries.

Engineering the Activity of a Template-Independent DNA Polymerase.

Enzymatic DNA writing technologies based on the template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) have the potential to advance DNA information storage. TdT is unique in its ability to synthesize single-stranded DNA de novo but has limitations, including catalytic inhibition by ribonucleotide presence and slower incorporation rates compared to replicative polymerases. We anticipate that protein engineering can improve, modulate, and tailor the enzyme's properties, but there is limited information on TdT sequence-structure-function relationships to facilitate rational approaches. Therefore, we developed an easily modifiable screening assay that can measure the TdT activity in high-throughput to evaluate large TdT mutant libraries. We demonstrated the assay's capabilities by engineering TdT mutants that exhibit both improved catalytic efficiency and improved activity in the presence of an inhibitor. We screened for and identified TdT variants with greater catalytic efficiency in both selectively incorporating deoxyribonucleotides and in the presence of deoxyribonucleotide/ribonucleotide mixes. Using this information from the screening assay, we rationally engineered other TdT homologues with the same properties. The emulsion-based assay we developed is, to the best of our knowledge, the first high-throughput screening assay that can measure TdT activity quantitatively and without the need for protein purification.

A massively parallel in vivo assay of TdT mutants yields variants with altered nucleotide insertion biases.

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA with random nucleotides. TdT's de novo DNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdT's intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants in high throughput. In our assay, a library of TdTs is encoded next to a CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9 and sgRNA, the target site is cut, allowing TdT to intercept the double strand break and add nucleotides. Each resulting insertion is sequenced alongside the identity of the TdT variant that generated it. Using this assay, 25,623 unique TdT variants, constructed by site-saturation mutagenesis at strategic positions, were profiled. This resulted in the isolation of several altered-bias TdTs that expanded the capabilities of our TdT-based DNA recording system, Cell History Recording by Ordered Insertion (CHYRON), by increasing the information density of recording through an unbiased TdT and achieving dual-channel recording of two distinct inducers (hypoxia and Wnt) through two differently biased TdTs. Select TdT variants were also tested in vitro , revealing concordance between each variant's in vitro bias and the in vivo bias determined from the multiplexed high throughput assay. Overall, our work, and the multiplex assay it features, should support the continued development of TdT-based DNA recorders, in vitro applications of TdT, and further study of the biology of TdT.

Evaluation of Asymptomatic Contralateral Foot Deformities Using the Tripod Index.

Background: The primary aim of this study was to determine the prevalence of asymptomatic pes planus and cavovarus foot deformities using the tripod index (TI). Methods: A retrospective study was conducted on 122 adult subjects over the age of 18 from January 2010 to December 2016 with symptomatic pes planus (n=78) or cavovarus (n=44) foot deformities. We subdivided both groups into subjects who presented with unilateral symptomatic deformities (pes planus unilateral symptomatic; cavovarus unilateral symptomatic) and bilateral symptomatic foot deformities (pes planus bilateral symptomatic feet and cavovarus bilateral symptomatic feet). The severity of TI was compared between sides. Results: The prevalence of asymptomatic pes planus and cavovarus foot deformities was 52% and 67.6%, respectively. Subjects with unilateral symptomatic foot deformities had significantly more severe TI values for the symptomatic cavovarus foot -98.96% (-288.89 to 0%) compared to asymptomatic cavovarus -67.41% (-270.59 to 14.71%) (p=0.015). Subjects with unilateral symptomatic pes planus deformity also had more severe TI on the symptomatic foot 57.49 (-9.38 to 141.67%) compared to the asymptomatic foot 30.43 (-51.52 to 119.23%) (p<0.01). Subjects with bilateral symptomatic foot deformities had no significant difference in severity of Tripod Index between feet. Conclusion: Although half of subjects with unilateral symptomatic deformities had a foot deformity on the contralateral side, the severity of deformity between symptomatic and asymptomatic feet was significantly different for both pes planus and cavovarus feet. Further studies should prospectively follow postoperative radiographs to determine whether a correction in foot alignment directly improves symptoms.Level of evidence: III.

Draft Genome Sequences of Thermophiles Isolated from Yates Shaft, a Deep-Subsurface Environment.

The whole-genome sequences of seven thermophiles that could grow at >55°C, but not at 37°C, were generated. These thermophilic bacteria will play a useful role as model microorganisms, and analyzing their genomes will help to understand the observed production of novel bioactive compounds, including thermozymes and macromolecules.

Illness-associated muscle weakness in dystroglycanopathies.

OBJECTIVE: To describe the phenomenon of acute illness-associated weakness (AIAW) in patients with dystroglycanopathy (DG), determine the frequency of this phenomenon in DGs, and compare it to the frequency in Duchenne-Becker muscular dystrophy (DBMD). METHODS: Patients enrolled in a DG natural history study provided medical history, including major illnesses or hospitalizations, at enrollment and annually. We noted a recurring syndrome of profound transient weakness in the setting of febrile illness. To determine the frequency of this phenomenon in the DG cohort and compare it to a cohort with another membrane-related muscular dystrophy, DBMD, we surveyed patients (e-survey tool), collecting demographics and information about episodes of sudden progression of weakness and events surrounding the episodes. RESULTS: Surveys were completed by 52 (56.6%) patients with DG and 51 (27.3%) patients with DBMD. AIAW was reported in 12 (23%) patients with DG and 2 (4%) patients with DBMD (odds ratio 7.35; 95% confidence interval 1.55, 34.77; p = 0.005). Altogether (history or survey), 21 patients with DG, with mutations in FKRP, FKTN, POMT1, POMT2, or POMGNT1, reported AIAW. These events typically occurred in children <7 years old, and the preceding illness usually included respiratory symptoms. In 10 (47.6%) patients with DG, AIAW preceded the diagnosis of muscular dystrophy. CONCLUSIONS: People with DG, across genotypes, can experience acute, transient weakness associated with a febrile illness, a phenomenon that rarely occurs in DBMD. The physiologic basis of this phenomenon is unknown. CLINICALTRIALSGOV IDENTIFIER: NCT00313677.