Zinc-finger nucleases facilitate in vivo integration of transgenes into the albumin locus

A group from the Children's Hospital of Philadelphia published a unique way to ameliorate hereditary bleeding disorders and enzyme deficiencies. Katherine A. High and her group utilized adeno-associated virus 8 (AAV8) to deliver a pair of zinc-finger nucleases (ZFNs) along with an interchangeable transgene to the liver. The transgene cassette is promoterless but is flanked by terminal regions homologous to the albumin gene. Upon ZFN cleavage, the transgene integrates with low efficiency, but under a very strong promoter, which leads to phenotype correction in a model of hemophilia A and hemophilia B. The correction of hemophilia A is particularly significant, since the gene encoding for factor VIII is larger than the AAV capacity. Thus, the most important advantage of this current method is that it increases AAV coding capacity by obviating the need of the promoter. In contrast to Mark Kay's recent study in Nature, this study did not observe any gene expression in the sole presence of an AAV encoding for the promoterless transgene. The authors noted some off-target effects, which might add up over time, given the long-term expression of ZFNs from an AAV episome.

Read the paper on the Blood journal website: http://www.bloodjournal.org/content/early/2015/08/20/blood-2014-12-615492.long?sso-checked=true

Parasites are targets for genome editing

There was a paper last week in Nature by the Striepen lab at the University of Georgia, showing that the well-known CRISPR system could be used to study an important human pathogen, Cryptosporodium parvum. The protozoan parasite causes diarrhea in children and in immunocompromised individuals (the image shows the parazites in the epithelial vacuoles of the intestine). Studying this parasite remains challenging due to lack of efficient propagation methods, simple animals models and molecular genetic manipulation techniques. Despite inefficient transfection, CRISPR/Cas9 along with a drug resistance cassette, rendered the parazites drug-resistant. Interestingly, non-homologous end-joining, a common error-prone DNA repair mechanism in higher organisms, is completely absent in Cryptosporodium. In contrast, homology-directed repair can lead to modifications in the genome when linked to Cas9 nuclease activity. The advance of this technique is that genetically modified parasites could be propagated and placed under selection pressure and in vivo. This would faciliate drug screening, drug target validation, but might also lead to the development of genetically modified, attenuated parasites for vaccination.

You can access the paper here: http://www.nature.com/nature/journal/v523/n7561/full/nature14651.html