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Using next-generation sequencing-based cancer gene panel analysis, several variants of unknown significance have been found in clinical cancer cases. One of VUS's main goals is the functional investigation of cultured cancer cell lines that harbor specific gene variants using genome editing. In human glioblastoma multiforme cell lines, we conducted comparative experiments to determine the most suitable editing conditions for the introduction of platelet-derived growth factor receptor alpha variants. The methods of transfection, methods of transfection, and cell sorting were all investigated to determine the editing quality in these GBM cell lines. Under our experimental conditions, the Cas9 RNP complex editing system demonstrated a higher editing quality than the Cas9 plasmid lipofection technique, making it the most effective method for single-nucleotide editing in human GBM cell lines.
Source link: https://doi.org/10.3390/ijms24010500
A TargeTron integration into selD, encoding the selenophosphate synthetase that is essential for specific incorporation of selenium into selenoproteins, results in a substantial rise defect and a global absence of selenium incorporation. The selD CRISPR mutation had a growth defect in protein-rich medium and mimicked a naturally inherited TargeTron selD mutant's phenotype. Our results indicate that Stickland metabolism may be a target for future antibiotic treatments and that the CRISPR-Cas9 system can deliver rapid and effective changes to the C.
Source link: https://doi.org/10.1038/s41598-017-15236-5
We present an effective genome editing strategy for bread wheat using CRISPR/Cas9 ribonucleoproteins. Starting from RNP preparation, the whole procedure takes only seven to nine weeks, with four to five independent mutants derived from 100 immature wheat embryos. The chance of off-target mutations in wheat cells is much smaller in RNP-mediated genome editing than in editing with CRISPR/Cas9 DNA, according to Deep sequencing results. The mutants obtained are utterly transgene free because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing.
Source link: https://doi.org/10.1038/ncomms14261
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