Base editing requires that the target sequence satisfy the protospacer adjacent motif requirement of the Cas9 domain and that the target nucleotide be located within the editing window of the base ...editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG protospacer adjacent motifs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4-5 nucleotides to up to ~8-9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing.
Pteropods have been nicknamed the “canary in the coal mine” for ocean acidification because they are predicted to be among the first organisms to be affected by changing ocean chemistry. This is due ...to their fragile, aragonitic shells and high abundances in polar and subpolar regions where the impacts of ocean acidification are most pronounced. For pteropods to be used most effectively as indicators of ocean acidification, the biotic and abiotic factors influencing their shell formation and dissolution in the modern ocean need to be quantified and understood. Here, we measured the shell condition (i.e., the degree to which a shell has dissolved) and shell characteristics, including size, number of whorls, shell thickness, and shell volume (i.e., amount of shell material) of nearly 50 specimens of the pteropod species Heliconoides inflatus sampled from a sediment trap in the Cariaco Basin, Venezuela, over an 11-month period. The shell condition of pteropods from sediment traps has the potential to be altered at three stages: (1) when the organisms are live in the water column associated with ocean acidification, (2) when organisms are dead in the water column associated with biotic decay of organic matter and/or abiotic dissolution associated with ocean acidification, and (3) when organisms are in the closed sediment trap cup associated with abiotic alteration by the preservation solution. Shell condition was assessed using two methods: the Limacina Dissolution Index (LDX) and the opacity method. The opacity method was found to capture changes in shell condition only in the early stages of dissolution, whereas the LDX recorded dissolution changes over a much larger range. Because the water in the Cariaco Basin is supersaturated with respect to aragonite year-round, we assume no dissolution occurred during life, and there is no evidence that shell condition deteriorated with the length of time in the sediment trap. Light microscope and scanning electron microscope (SEM) images show the majority of alteration happened to dead pteropods while in the water column associated with the decay of organic matter. The most altered shells occurred in samples collected in September and October when water temperatures were warmest and when the amount of organic matter degradation, both within the shells of dead specimens and in the water column, was likely to have been the greatest. The hydrographic and chemical properties of the Cariaco Basin vary seasonally due to the movement of the Intertropical Convergence Zone (ITCZ). Shells of H. inflatus varied in size, number of whorls, and thickness throughout the year. There was not a strong correlation between the number of whorls and the shell diameter, suggesting that shell growth is plastic. H. inflatus formed shells that were 40 % thicker and 20 % larger in diameter during nutrient-rich, upwelling times when food supply was abundant, indicating that shell growth in this aragonite-supersaturated basin is controlled by food availability. This study produces a baseline dataset of the variability in shell characteristics of H. inflatus pteropods in the Cariaco Basin and documents the controls on alteration of specimens captured via sediment traps. The methodology outlined for assessing shell parameters establishes a protocol for generating similar baseline records for pteropod populations globally.
The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the ...DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.
The dissolution of the delicate shells of sea butterflies, or pteropods, has epitomised discussions regarding ecosystem vulnerability to ocean acidification over the last decade. However, a recent ...demonstration that the organic coating of the shell, the periostracum, is effective in inhibiting dissolution suggests that pteropod shells may not be as susceptible to ocean acidification as previously thought. Here we use micro-CT technology to show how, despite losing the entire thickness of the original shell in localised areas, specimens of polar species Limacina helicina maintain shell integrity by thickening the inner shell wall. One specimen collected within Fram Strait with a history of mechanical and dissolution damage generated four times the thickness of the original shell in repair material. The ability of pteropods to repair and maintain their shells, despite progressive loss, demonstrates a further resilience of these organisms to ocean acidification but at a likely metabolic cost.
The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or ...activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.
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•Cas9 can be circularly permuted (Cas9-CP) for optimized fusion protein construction•Cas9 circular permutation enables the engineering of protease-activated ProCas9s•ProCas9s can sense and respond to protease activity, such as during viral infection•Cas9-CPs provide a toolkit of safer and more efficient genome-modifying enzymes
Programmable Cas9 variants with improved functionality are generated by protein engineering—including versions that work as protease sensors responding to viral infection.
Extracellular vesicles (EVs) enclose mRNA derived from their cell of origin and are considered a source of potential biomarkers. We examined urinary EV mRNA from individuals with diabetic kidney ...disease (DKD), chronic kidney disease, type 2 diabetes (T2DM), and obese and healthy controls to determine if such biomarkers had the potential to classify kidney disease and predict patients at higher risk of renal function decline.
A total of 242 participants enrolled in this study. Urinary EV mRNA from all subjects were isolated by a filter-based platform, and the expression of 8 target genes were determined by quantitative polymerase chain reaction (qPCR). Changes in estimated glomerular filtration rate (eGFR) in 161 T2DM patients were evaluated for 2 consecutive years and compared with EV RNA profiles at baseline.
We observe that mild and severe DKD groups show a significant 3.2- and -4.4-fold increase in UMOD compared to healthy controls and expression increases linearly from healthy, diabetic, and DKD subjects. UMOD expression is significantly correlated to albumin creatinine ratio (ACR), eGFR, and HbA1c. Using linear discriminant analyses with mRNA from severe DKD and T2DM as training data, a multi-gene signature classified DKD and -non-DKD with a sensitivity of 93% and specificity of 73% with area under the receiver operating characteristic (ROC) curve (AUC) = 0.90. Although 6% of T2DM were determined to have a > 80% posterior probability of developing DKD based on this mRNA profile, eGFR changes observed within the 2-year follow-up did not reveal a decline in kidney function.
Urinary EV UMOD mRNA levels are progressively elevated from T2DM to DKD groups and correlate with widely used eGFR and ACR diagnostic criteria. An EV mRNA signature could identify DKD with greater than 90% sensitivity and 70% specificity.
The viewpoint of the terminally ill child at the time of an end-of-life decision has not been formally investigated. We identified the preferences of children and adolescents with advanced cancer ...about their end-of-life care and the factors that influenced their decisions.
Pediatric patients 10 or more years of age were interviewed within 7 days of participating in one of the following three end-of-life decisions: enrollment onto a phase I trial (n = 7), adoption of a do not resuscitate order (n = 5), or initiation of terminal care (n = 8). The patient, a parent, and the primary pediatric oncologist were interviewed separately by using open-ended interview questions.
Twenty patients, aged 10 to 20 years (mean, 17 years and 4 months), with a refractory solid tumor (n = 12), brain tumor (n = 4), or leukemia (n = 4) participated. Eighteen patients (90%) accurately recalled all of their treatment options and identified their own death as a consequence of their decision. The factors that were most frequently identified included the following: for patients, caring about others (n = 19 patients); for parents, the child's preferences (n = 18 parents); and for physicians, the patient's prognosis and comorbid conditions (n = 14 physicians).
These children and adolescents with advanced cancer realized that they were involved in an end-of-life decision, understood the consequences of their decision, and were capable of participating in a complex decision process involving risks to themselves and others. The decision factors most frequently reported by patients were relationship based; this finding is contrary to existing developmental theories.
When a child's cancer progresses beyond current treatment capability, the parents are likely to participate in noncurative treatment decision making. One factor that helps parents to make these ...decisions and remain satisfied with them afterward is deciding as they believe a good parent would decide. Because being a good parent to a child with incurable cancer has not been formally defined, we conducted a descriptive study to develop such a definition.
In face-to-face interviews, 62 parents who had made one of three decisions (enrollment on a phase I study, do not resuscitate status, or terminal care) for 58 patients responded to two open-ended questions about the definition of a good parent and about how clinicians could help them fulfill this role. For semantic content analysis of the interviews, a rater panel trained in this method independently coded all responses. Inter-rater reliability was excellent.
Among the aspects of the definition qualitatively identified were making informed, unselfish decisions in the child's best interest, remaining at the child's side, showing the child that he is cherished, teaching the child to make good decisions, advocating for the child with the staff, and promoting the child's health. We also identified 15 clinician strategies that help parents be a part of making these decisions on behalf of a child with advanced cancer.
The definition and the strategies may be used to guide clinicians in helping parents fulfill the good parent role and take comfort afterward in having acted as a good parent.
Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited ...experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.
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