CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence (‘guide sequence’) and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or ‘MP’) incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.
It is still open to question which method is the best for quantifying organic carbon (OC) and inorganic carbon (IC) in soils containing dolomite. The aims of this study were (1) to compare the accuracy of a novel thermal gradient (ThG), the classical calcimeter (CALC) and the loss-on-ignition (LOI) methods on a reference sample set with known proportions of OC present as soil organic matter (SOM) and IC present as dolomite and (2) to compare the results of the different methods on a set of soil samples with different dolomite and SOM contents. The CALC and LOI methods rely on separate quantification or removal of IC by acid or heat, whereas IC and OC can be quantified in a single run by the ThG analysis. The ThG method was the most accurate method for the reference sample set, especially when dolomite contents were high. On the soil sample set, the ThG and CALC methods performed equally well, but only when two outliers were eliminated. The LOI method was not satisfactory for either sample set. Overall, ThG was the most reliable method for measuring IC and OC in dolomite-containing samples over a wide range of concentrations, but the more widely used CALC method was also acceptable.
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