Biology subjects
Publications and source records attributed to Li, s..
Withdrawal statementThe authors have withdrawn this manuscript because of a duplicate posting of a preprint on arXiv. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The original preprint can be found at arXiv:2601.05648
The novel duck reovirus (NDRV) disease presents a significant threat to the poultry industry due to the absence of effective therapeutic measures. As a result, there is an urgent need to develop innovative rapid diagnostic methods for early virus detection. In this study, we developed a Rapid Visual CRISPR Assay to detect the NDRV S3 gene using novel Cas12a orthologs. Specifically, we compared the performance of two candidates, Gs12-16 and Gs12-18, in detecting the NDRV S3 gene to identify a highly sensitive and efficient CRISPR-based diagnostic method. Our results demonstrated that both Gs12-16 and Gs12-18 exhibited strong cis- and trans-cleavage activities for classical "TTTV" protospacer adjacent motif (PAM)-containing targets in vitro, although they required different reaction temperatures. Notably, Gs12-18 showed relatively higher activity for dsDNA targets compared to Gs12-16, indicating that Gs12-18 is more suitable for CRISPR-based nucleic acid detection applications. To leverage these properties, we integrated Gs12-18 with loop-mediated isothermal amplification (LAMP) technology to establish a LAMP-CRISPR/Gs12-18-mediated method for detecting the NDRV S3 gene. This approach enables highly sensitive and visually detectable on-site identification of the NDRV S3 gene, achieving a sensitivity of 38 copies per reaction. Our LAMP-CRISPR/Gs12-18-based method can be utilized for highly sensitive detection of NDRV nucleic acids.
RAS genes are commonly mutated in cancers yet despite many possible mutations, cancers have a tropism towards a specific subset. As driver mutations, these patterns ostensibly originate from normal cells. High oncogenic RAS activity causes oncogenic stress and different oncogenic mutations can impart different levels of activity. Here we show that changing rare codons to common in the murine Kras gene to increase translation shifts tumors induced by the carcinogen urethane from arising from canonical Q61 to biochemically less active G12 Kras driver mutations, despite the carcinogen still being biased towards generating Q61 mutations. Loss of p53 to blunt oncogenic stress partially reversed this effect, restoring Q61 mutations. Finally, transcriptional analysis revealed similar signaling amongst tumors driven by different mutations and Kras alleles. These finding suggest that the RAS mutation tropism of urethane is largely product of selection in normal cells for mutations promoting proliferation without causing oncogenic stress. Impact statementThe bias towards specific Kras driver mutations during urethane carcinogenesis appears to arise predominantly from the selection of a narrow window of oncogenic signaling in normal cells.