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Genetic Engineering Publications - GEG Tech top picks
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BigField GEG Tech's insight:
The fusion Cas9/FokI enhances specificity of CRISPR system without reduce the efficiency. In human cells, the authors shown that this fusion allows the modification of target DNA sites with a 140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases'.
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Scoop.it!
BigField GEG Tech's insight:
In this chapter, the scientists describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. They show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.
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Here, the scientists report the development of DNA aptamers that bind to the target molecules, with high affinity and specificity to the FokI. DNA aptamers were selected in six rounds of systematic evolution of ligands by exponential enrichment. They find two aptamers which showed high binding affinity to FokI, resistance to nuclease activity itself and did not inhibit nuclease activity. These aptamers could be useful for genome editing applications such as controlled delivery of SSNs.