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

Split-Cas9 design and its implications for AAV-mediated CRISPR delivery

Wright AV et al, PNAS 2015,
www.pnas.org/cgi/doi/10.1073/pnas.1501698112, copyright: PNAS

In their paper, Wright et al. separated the Cas9 enzyme into two distinct parts, a nuclease lobe and an α-helicase lobe. The two different polypeptides were shown to be brought together by the guide RNA, reconstituting the active CRISPR complex. One of the most important applications of this system is that two smaller regions of Cas9 could be cloned into AAV, overcoming capacity limitations of this vector. Moreover, there would be more room for larger or inducible promoters to spatiotemporally limit Cas9 expression. Inducible dimerization domains may also be applied to regulate expression. Readers should be aware that this split Cas9 sytem seems to be less effective than wild-type Cas9, WT Cas9 generated indels in HEK293T cells with around 22% frequency, but this was only 0.6% with split-Cas9 (synchronized cells showed slightly increased indel formation). This warrants further optimization, but having the Cas9 on two different polypeptides definitely has advantages. The lower indel rate compared to WT Cas9 is in accordance with the split Cas9 from the Zetsche B (http://goo.gl/J5Ogqu) paper. The difference in the Wright and the Zetsche paper is that in the latter, the Cas9 is cut in half (to obtain an N- and a C-terminal domain), whereas in the Wright paper the nuclease lobe contains a short N-terminal part and a long C-terminal part (with a short linker in between) and the α-helicase lobe is consisted of 'middle' amino acids. The Wright strategy split Cas9 is able to dimerize spontaneously in the presence of guide RNA, but the Zetsche split Cas9 needs chemically inducible dimerization domains for function.  

Original paper: Wright AV, Sternberg SH, Taylor DW, Staahl BT, Bardales JA, Kornfeld JE, Doudna JA. Rational design of a split-Cas9 enzyme complex. Proc Natl Acad Sci U S A. 2015 Feb 23. pii: 201501698a, http://www.pnas.org/content/early/2015/02/18/1501698112.long