CRISPR-Cas systems are prokaryotic immune systems that have proliferated widely not only in bacteria and archaea, but also much more recently, in human biological research and applications. Much work to date has utilized synthetic sgRNAs along with the CRISPR nuclease Cas9, but the discovery of array-processing nucleases now allows the use of more compact, natural CRISPR arrays in heterologous hosts, in addition to organisms with endogenous systems. Unfortunately, the construction of multiplex natural CRISPR arrays remains technically challenging, expensive, and/or time-consuming. This limitation hampers research involving natural CRISPR arrays in both native and heterologous hosts. To address this problem, we present a method to assemble CRISPR arrays that is simple, rapid, affordable, and highly scalable-we assembled 9-spacer arrays with 1 day's worth of work. We used this method to harness the endogenous CRISPR-Cas system of the highly competent bacterium Acinetobacter baylyi, showing that while single spacers are not always completely effective at blocking DNA acquisition through natural competence, multiplex natural CRISPR arrays enable both nearly complete DNA exclusion and genome editing, including with multiple targets for both. In addition to demonstrating a CRISPR array assembly method that will benefit a variety of applications, we also find a potential bet-hedging strategy for balancing CRISPR defense versus DNA acquisition in naturally competent A. baylyi.

Objectives

It has been suggested that statins exert potential anti-tumor effects. The relationship between statin use and outcomes in pancreatic cancer is controversial. We hypothesized that statin use at baseline would impact survival among patients with early-stage pancreatic cancer and that the effect might vary by individual statin agent.

Methods

We conducted a retrospective cohort study on data from an integrated healthcare system. We included patients with pancreatic cancer stage I-IIb who underwent resection for curative intent between January 2005 and January 2011. Baseline statin use was characterized as any prior use as well as active use of either simvastatin or lovastatin. Intensity of exposure was calculated as average daily dose prior to surgery. Overall and disease-free survival was assessed from surgery until the end of study (April 2014). We used the Kaplan-Meier method and Cox proportional hazards regression to evaluate the impact of baseline statin use on survival, adjusting for age, sex, Charlson comorbidity score, resection margin, disease stage, and receipt of adjuvant chemotherapy.

Results

Among 226 patients, 71 (31.4%) had prior simvastatin use and 27 (11.9%) had prior lovastatin use at baseline. Prior simvastatin but not lovastatin use was associated with improved survival (median 28.5 months (95% confidence limit (CL) 20.8, 38.4) for simvastatin vs. 12.9 months (9.6, 15.5) for lovastatin vs. 16.5 months (14.1, 18.9) for non-statin users; log-rank P=0.0035). In Cox regression, active simvastatin use was independently associated with reduced risk for mortality (adjusted hazard ratio (HR) 0.56 (95% CL 0.38, 0.83), P=0.004) and risk for recurrence (adjusted HR 0.61 (0.41, 0.89), P=0.01). Survival improved significantly among patients who received moderate-high-intensity (median 42.1 months (24.0,52.7)) doses compared with those who received low-intensity doses of simvastatin (median 14.1 months (8.6, 23.8), log-rank P=0.03).

Conclusions

The effects of statins varied by agent and dose. Active use of moderate-high-dose simvastatin at baseline was associated with improved overall and disease-free survival among patients undergoing resection for pancreatic cancer.

Synthetic biology has developed sophisticated cellular biosensors to detect and respond to human disease. However, biosensors have not yet been engineered to detect specific extracellular DNA sequences and mutations. Here, we engineered naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells, organoids, and tumors. We characterized the functionality of the biosensors in vitro with coculture assays and then validated them in vivo with sensor bacteria delivered to mice harboring colorectal tumors. We observed horizontal gene transfer from the tumor to the sensor bacteria in our mouse model of CRC. This cellular assay for targeted, CRISPR-discriminated horizontal gene transfer (CATCH) enables the biodetection of specific cell-free DNA.

Background and aims

Childhood acute lymphoblastic leukemia (ALL) is a biologically heterogeneous disease, and mutations in the KRAS and NRAS oncogenes are present at diagnosis in about one-fifth of cases. Ras mutations were previously associated with environmental exposures in leukemias as well as in many other cancer types. This study examined whether Ras mutation could define a unique etiologic group of childhood ALL associated with tobacco smoke, a well-established mutagen and carcinogen.

Methods

We included 670 children with ALL enrolled in a case-control study in California (1995-2013), including 50.6% Latinos. Parental and child exposure to tobacco smoke was obtained from interviews. Sanger sequencing was used to detect the common KRAS and NRAS hotspot mutations in diagnostic bone marrow DNA. ALL cases were also characterized for common chromosome abnormalities. In case-case analyses, logistic regression analyses were used to estimate odds ratios to describe the association between tobacco smoke exposure and childhood ALL with Ras mutations.

Results

KRAS or NRAS mutations were detected in ∼18% of children diagnosed with ALL. Ras mutations were more common among Latino cases compared with non-Latino whites and in high-hyperdiploid ALL. No associations were observed between parental smoking or child's passive exposure to smoke and Ras positive ALL.

Conclusions

The apparent lack of association between tobacco smoke and Ras mutation in childhood ALL suggests that Ras mutations do not specifically define a tobacco-related etiologic pathway. Reasons for racial and ethnic differences in ALL are not well understood and could reflect differences in etiology that warrant further examination.