Optical control of genome editing

The group of Moritoshi Sato (University of Tokyo) reported a novel, photoactivatable Cas9 variant (paCas9) for spatiotemporally regulated genome editing. Researchers made a split Cas9, with fragments fused to 'Magnet' dimerization domains. Upon blue light, Magnet domains bring together Cas9 components and reconstitute genome editing activity. The system is a little bit less effective than wtCas9, but it's activation is reversible and could be controlled in space. The authors provided evidence that the system works also with Cas9-nicakse and also with dead Cas9 (dCas9) for reversible transcriptional inhibition. The current constructs are small enough to be cloned into AAV. It represents an alternative to doxycycline regulated or rapamyicin-inducible Cas9 systems. Check out the paper here: http://goo.gl/vw5H1Z

Want to design a CRISPR for genome editing? Here are the PAM sites you need to keep in mind:

The PAM site is the only sequence requirement when designing a specific guide RNA for the CRISPR, i.e. only sites in the genomic DNA next to a specific PAM motif could be targeted and edited. The PAM site is in the genomic DNA, not in the guide RNA. The presence or absence of this motif next to your favorite sequence is one of the major limitations of the CRISPR/Cas9 system. If a Cas9 would be utilized to cleave an allele with a mutation, the mutation must be next (within preferably 10nt) to a PAM site.

(The PAM site is needed for the bacterial CRISPR system to discriminate the bacterial and viral genome, since the PAM site is absent from the former but present in the latter.)

Here are the PAM sites for the currently used Cas9 enzymes, check if you find a good one next to your sequence of interest:

R: A or G, W: A or T
*Be careful with non-canonical PAM sites, at some sites there might be no activity
**Not yet available, will be available soon

1. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013 Feb 15;339(6121):819-23. doi: 10.1126/science.1231143.
2. Zhang Y, Ge X, Yang F, Zhang L, Zheng J, Tan X, Jin ZB, Qu J, Gu F. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells. Sci Rep. 2014 Jun 23;4:5405. doi: 10.1038/srep05405.
3. Friedland AE, Sousa A, Collins M. et al. S. aureus Cas9: and alternative Cas9 for genome editing applications, Editas Medicine, http://paperzz.com/doc/3029869/read-more---editas-medicine
4. Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15644-9. doi: 10.1073/pnas.1313587110.
5. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 2011 Nov;39(21):9275-82. doi: 10.1093/nar/gkr606.

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