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.

The development of immunological memory in bacteria 

CRISPR mediated bacterial immunity, with permission
from NPG, from Yosef I et al, Nature 2015

Two papers published last week in Nature describe the amazing development of bacterial immunological memory. Previously it has been thought that in contrast to vertebrates, bacteria can not remember previous infections. Now it is obvious that the original function of CRISPR is to fight against invading viruses by cleaving specific parts of the viral genome. During an infection, part of the viral genome is stored in the bacterial genome as a library, from which a matching sequence can be utilized upon subsequent infection. How this happens remained elusive until recently, now it is shown that components of the CRISPR system (Cas1, Cas2, Csn1, Cas9 and tracrRNA) act in concert to find appropriate target regions in the viral genome, and insert a copy into the bacterial genome. Finally it makes sense, why there is a so-called PAM site. This is a very short motif adjacent to the recognition region, which is an absolute sequence requirement of the system (e.g. this is NGG for the S. pyogenes CRISPR system). Most importantly, when pasted into the bacterial genome, there is no PAM site next to the sequence. So that is why bacteria do not attack themselves, since the PAM site is only in the virus, leading to cleavage. That is how bacteria evade autoimmunity. Smart!

Check out the original papers here: http://goo.gl/kz2X1V