CRISPR Digest #9

The Economist magazine had a field day in their issue of October 17th 2015: in their article, “No pig in a poke” – with one of the subtitles being “Go the whole hog” – they describe the latest findings by Yang et al. (2015) in which they used CRISPR/Cas9 genome-engineering technology to remove potentially contagious virus sequences from pig genomes. These DNA sequences are called porcine endogenous retroviruses (PERVs for short) and could move into the human genome if, for example, a pig heart were transplanted into a human. In addition to the problem of immune rejection of the organ, therefore, there is also the risk of infection with porcine viruses/DNA sequences. The Economist concluded that, “The existence of PERVs, then, has been one of the main obstacles to transplanting pig organs into people”. In this proof-of-concept paper by Yang et al. the scientists managed to remove DNA sequences necessary for the PERVs to move from the pig genome into the human genome. However, this was done in cell culture and not in whole pigs, “but this is still a striking result” despite the fact that “a single paper does not a new medical procedure make”. [I did not realise quite how poetic the writers at The Economist can be.]

In other news, more basic research on CRISPR has revealed that the Cas9 enzyme – the protein that makes the cut in DNA – of a bacterial species called Neisseria meningitidis can also cut single strands of DNA (Zhang et al., 2015), in addition to its expected role of cutting double strands of DNA. This is of interest because it allows a wider range of applications, especially considering that N. meningitidis Cas9 is considerably smaller, and therefore easier to handle from an experimental point of view, than Streptococcus pyogenes Cas9, which is currently the most commonly used Cas9 version.

While many research groups are working on understanding new mechanisms of gene editing and how various Cas9 variants function in detail, the group of Jennifer Doudna published a paper in Nature this week outlining how E. coli acquire foreign DNA and integrate it into their genomes’ CRISPR arrays (Nuñez et al., 2015). They studied the crystal structure of the complex of the Cas1 and Cas2 proteins, which are well conserved between bacterial species, and the DNA to be integrated:

X-ray crystal structure of E. coli Cas1-Cas2 in complex with double-stranded DNA - image copied directly from Nuñez et al., 2015

X-ray crystal structure of E. coli Cas1-Cas2 (blue/green and yellow) in complex with double-stranded DNA (red) – image copied directly from Nuñez et al., 2015

In particular, Nuñez et al. found that the Cas1-Cas2 complex acts as a molecular ruler to determine the length of DNA that is incorporated into the E. coli genome. Furthermore, they demonstrate that Cas1 can pry open the ends of the DNA, as seen in the image above, so that Cas2 – which is catalytically active – can gain access to the reactive ends of the DNA. So here is yet another example of how the structure and function of proteins are intricately connected and often knowledge of one can enhance knowledge of the other.

References:

Nuñez JK, Harrington LB, Kranzusch PJ, Engelman AN, Doudna JA (2015) Foreign DNA capture during CRISPR–Cas adaptive immunity. Nature advance online publication

Yang L, Güell M, Niu D, George H, Lesha E, Grishin D, Aach J, Shrock E, Xu W, Poci J, Cortazio R, Wilkinson RA, Fishman JA, Church G (2015) Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science

Zhang Y, Rajan R, Seifert HS, Mondragón A, Sontheimer Erik J (2015) DNase H Activity of Neisseria meningitidis Cas9. Molecular Cell 60: 242-255

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