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CRISPR/Cas9 ribonucleoprotein (RNP) complexes are promising biological tools with diverse biomedical applications. However, to date there are no efficient methods that can produce these proteins at large scales and low cost. Here, we present a streamlined method for direct production of Cas9 RNPs from Escherichia coli by co-expression of Cas9 and the target-specific single-guided RNAs. Harnessing an ultrahigh-affinity CL7/Im7 purification system recently developed we achieve one-step purification of the self-assembling CRISPR/Cas RNPs, including the commonly used Cas9 and Cas12a, within half a day and with a ~fourfold higher yield than incumbent methods. The prepared Cas RNPs show remarkable stability in the absence of RNase inhibitors, as well as profound gene-editing efficiency in vitro and in vivo. Our method is convenient, cost-effective, and can be used to prepare other CRISPR/Cas RNPs., Competing Interests: W.Q.L., L.X.M., and Y.L. have applied a provisional patent on direct production of CRISPR/Cas RNPs from E. coli, which partly presented in this paper. The remaining authors declare no competing interests.

Brackish water samples from Lake Pontchartrain in Louisiana were assessed for the presence of pathogenic amoeba Naegleria fowleri, which causes primary amoebic meningoencephalitis (PAM). In our study, quantitative polymerase chain reaction (qPCR) methods were used to determine N. fowleri, E. coli, and enterococci in water collected from Lake Pontchartrain. N. fowleri target sequence was detected in 35.4% (56/158) of the water samples from ten sites around the lake. Statistically significant positive correlations between N. fowleri concentration and water temperature as well as E. coli (qPCR) were observed. Multiple linear regression (MLR) model shows seasonal factor (summer or winter) has significant effect on the concentration of N. fowleri, E. coli and enterococci (qPCR) concentration. Significant positive relationships between E. coli and enterococci was observed from both qPCR (r=0.25) and culture based method (r=0.54). Meanwhile, significant positive correlation between qPCR and culture based methods for enterococci concentration was observed (r=0.33). In our study, water temperature and E. coli concentration were indicative of N. fowleri concentrations in brackish water environment. Future research is needed to determine whether sediment is a source of N. fowleri found in the water column., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Water quality in Lake Pontchartrain was deteriorating and recreational activities along the beach were restricted by the end of the 20th Century. A microbial source tracking (MST) study was conducted to determine the fecal contamination sources at public beach of the lake, so that effective pollution control strategies can be developed. Water samples were collected over an eight-month period at ten locations along the lake in 2016 and 2017. E. coli and Enterococcus were detected in 90.6% (culture) and 97.5% (qPCR), 95.8% (culture) and 91.8% (qPCR) of water samples from all sampling sites, respectively. Significant positive relationship between E. coli and Enterococcus results was observed for both qPCR and culture methods. HF183 marker was detected in 94.3% water samples (149 of 158), with concentrations ranging from 29.0 to 6073.5 GC/100 ml and from 129.8 to 38,465.6 GC/100 ml in summer and winter, respectively. The results also indicate that significant rainfall events have the potential to supply considerable loads of fecal bacteria to lake waters. Further research is needed to determine the contribution of other animals to fecal contamination in the region. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]