CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRN...CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.展开更多
Alkaline and ultrasonic sludge disintegration can be used as the pretreatment of waste activated sludge （WAS） to promote the subsequent anaerobic or aerobic digestion. In this study, different combinations of these ...Alkaline and ultrasonic sludge disintegration can be used as the pretreatment of waste activated sludge （WAS） to promote the subsequent anaerobic or aerobic digestion. In this study, different combinations of these two methods were investigated. The evaluation was based on the quantity of soluble chemical oxygen demand （SCOD） in the pretreated sludge as well as the degradation of organic matter in the subsequent aerobic digestion. For WAS samples with combined pretreatment, the released COD levels were higher than those with ultrasonic or alkaline pretreatment alone. When combined with the ultrasonic treatment, NaOH treatment was more efficient than Ca（OH）2 for WAS solubilization. The COD levels released in various sequential options of combined NaOH and ultrasonic treatments were in the the following descending order： simultaneous treatment 〉 NaOH treatment followed by ultrasonic treatment 〉 ultrasonic treatment followed by NaOH treatment. For simultaneous treatment, low NaOH dosage （100 g/kg dry solid）, short duration （30 min） of NaOH treatment, and low ultrasonic specific energy （7500 kJ/kg dry solid） were suitable for sludge disintegration. Using combined NaOH and ultrasonic pretreatment with optimal parameters, the degradation efficiency of organic matter was increased from 38.0% to 50.7%, which is much higher than that with ultrasonic （42.5%） or with NaOH pretreatment （43.5%） in the subsequent aerobic digestion at the same retention time.展开更多
文摘CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.
基金supported by the China NationalEleventh Five-Year Scientific and Technical Support Plan(No. 2006BAC02A18)
文摘Alkaline and ultrasonic sludge disintegration can be used as the pretreatment of waste activated sludge （WAS） to promote the subsequent anaerobic or aerobic digestion. In this study, different combinations of these two methods were investigated. The evaluation was based on the quantity of soluble chemical oxygen demand （SCOD） in the pretreated sludge as well as the degradation of organic matter in the subsequent aerobic digestion. For WAS samples with combined pretreatment, the released COD levels were higher than those with ultrasonic or alkaline pretreatment alone. When combined with the ultrasonic treatment, NaOH treatment was more efficient than Ca（OH）2 for WAS solubilization. The COD levels released in various sequential options of combined NaOH and ultrasonic treatments were in the the following descending order： simultaneous treatment 〉 NaOH treatment followed by ultrasonic treatment 〉 ultrasonic treatment followed by NaOH treatment. For simultaneous treatment, low NaOH dosage （100 g/kg dry solid）, short duration （30 min） of NaOH treatment, and low ultrasonic specific energy （7500 kJ/kg dry solid） were suitable for sludge disintegration. Using combined NaOH and ultrasonic pretreatment with optimal parameters, the degradation efficiency of organic matter was increased from 38.0% to 50.7%, which is much higher than that with ultrasonic （42.5%） or with NaOH pretreatment （43.5%） in the subsequent aerobic digestion at the same retention time.
