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Genetic Engineering Publications - GEG Tech top picks
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BigField GEG Tech's insight:
In this work, the scientists show that using phosphorothioate-modified oligonucleotides strongly enhances genomeediting efficiency of single-stranded oligonucleotide donors in cultured cells. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. 
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This CRISPR Cas9 sgRNA tracrRNA modification study shows that it is possible to improve quality of sgRNA libraries with changes in the Cas9 binding region
BigField GEG Tech's insight:
One study (Chen, et al.) with an inactive Cas9 nuclease has shown that sequence modifications to the constant region improved Cas9 binding significantly. However, since an inactive Cas9 mutant was used in these studies, it wasn’t clear if the changes would actually increase the rate or the knockout efficiency of the active CRISPR system, and further, if they did, the effect that they would have on results of CRISPR-based pooled genetic screens. Here, the scientists initiated a study to address these points.
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In this study two strategies were used to increase the precise genetic modification in rats. Scr7, a DNA ligase IV inhibitor, first identified as an anti-cancer compound, and considered as a potential NHEJ inhibitor, was used to increase the HR-mediated precise genetic modification. Meanwhile, the Cas9 protein instead of mRNA was used to save the mRNA to protein translation step to improve the precise modification efficiency. The Fabp2 and Dbndd1 loci were selected to knockin Cre and CreERT2, respectively. These results showed that both Scr7 and Cas9 protein can increase the precise modification.