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