Research and publish the best content.
Get Started for FREE
Sign up with Facebook Sign up with X
I don't have a Facebook or a X account
Already have an account: Login
Genetic Engineering Publications - GEG Tech top picks
36.6K views |
+0 today
 Your new post is loading...
Your new post is loading...
Scoop.it!
BigField GEG Tech's insight:
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. 
Scoop.it!
BigField GEG Tech's insight:
In this study, the authors show that a site specific double strand break (DSB) generated both in the genome and the donor plasmid using the CRISPR-Cas9 system can be efficiently used to target ∼5 kb plasmids into mammalian genomes via nonhomologous end joining (NHEJ). They were able to achieve efficiencies of up to 0.17% in HEK293 cells and 0.45% in CHO cells.
Dominique Blanchard's comment,
May 13, 2015 8:46 AM
I'm going to read this paper because It will greatly impact owners of homologous recombination related patents.
Scoop.it!
BigField GEG Tech's insight:
The authors targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7 to promote HDR at the expense of NHEJ. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold.
|
Scoop.it!
A gain-of-function mutation in a sodium/potassium pump renders cells resistant to a small-molecule drug and provides an efficient coselection strategy to enrich for CRISPR-induced mutations at an independent locus.
BigField GEG Tech's insight:
Here, the scientists designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems.
Scoop.it!
BigField GEG Tech's insight:
Genome editing using designer nucleases such as TALENs or the CRISPR-Cas9 system is hampered by a lack of methods to detect and quantify the products. Here the authors present GEF-dPCR, a droplet-based digital PCR method for assessing gene-editing frequencies.
Scoop.it!
BigField GEG Tech's insight:
The authors demonstrated that suppressed the NHEJ key molecules such as KU70 or DNA ligase IV promotes the efficiency of HDR 4–5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines.
Scoop.it!
BigField GEG Tech's insight:
As human Rap1, like its mouse and unicellular orthologs, affects gene expression, the authors propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres.
|
Despite high hopes and high investment in CRISPR-Cas9 gene editing, scientists still have a lot to learn about how it works in humans.