Crystal structure of Staphylococcus aureus Cas9 has been revealed

Feng Zhang (MIT, Cambridge) and a group from Tokyo provided 2.7 Å resolution structures for the smaller Cas9 variant from S. aureus. This Cas9 gained attention owing to it's size compatible with adeno-associated viral vectors. The PAM site (DNA recognition site) of this variant is 'TTGAAT' or 'TTGGGT', which is however more complex than the S. pyogenes 'NGG' PAM site. Interestingly, the S. aureus and the S. pyogenes Cas9 share
only 17% sequence identity, but have similar overall domain organization. Structural information enabled rational design of S. aureus Cas9-based transcriptional activators and split Cas9 design.





Link to paper: http://www.cell.com/cell/abstract/S0092-8674(15)01020-X

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

Parasites are targets for genome editing

There was a paper last week in Nature by the Striepen lab at the University of Georgia, showing that the well-known CRISPR system could be used to study an important human pathogen, Cryptosporodium parvum. The protozoan parasite causes diarrhea in children and in immunocompromised individuals (the image shows the parazites in the epithelial vacuoles of the intestine). Studying this parasite remains challenging due to lack of efficient propagation methods, simple animals models and molecular genetic manipulation techniques. Despite inefficient transfection, CRISPR/Cas9 along with a drug resistance cassette, rendered the parazites drug-resistant. Interestingly, non-homologous end-joining, a common error-prone DNA repair mechanism in higher organisms, is completely absent in Cryptosporodium. In contrast, homology-directed repair can lead to modifications in the genome when linked to Cas9 nuclease activity. The advance of this technique is that genetically modified parasites could be propagated and placed under selection pressure and in vivo. This would faciliate drug screening, drug target validation, but might also lead to the development of genetically modified, attenuated parasites for vaccination.

You can access the paper here: http://www.nature.com/nature/journal/v523/n7561/full/nature14651.html

New high-throughput analysis reveals parameters affecting guideRNA efficiency

A recent report in Nature Methods by one of the leading geneticist, George Church, unveils factors that affect genome editing efficiency with CRISPR. The group from the Wyss Institute and Harvard Medical School employed a library-on-library approach to figure out why certain gRNAs are more effective than others. They carried out two separate experiments; first they transfected cells with a target lentiviral library as well as a gRNA plasmid library, which enabled the scientists to analyze how a certain sequence affect genome editing rates. Not quite surprising, there was a massive variation among different gRNAs at different targets, but the most important base was found to be the PAM adjacent base, with G being the most efficient and T least preferred. Next, they transfected the gRNA plasmid library into 293T cells and looked at endogenous genome editing. This experiment allowed to analyze the effect of epigenetic parameters on genome editing efficiency. Indeed, there was a robust correlation between DN-ase I hypersensitive sites (i.e. more open chromatin, transcriptionally active regions) and editing efficiency. Finally, it is interesting to note that there was no tangible relationship between gRNA effectiveness and off-target activity, moreover some of the most active gRNAs had the least off-target effects.

The group made a prediction algorithm available
online: https://crispr.med.harvard.edu/sgRNAScorer/

Protospacer Workbench - an offline software for for rapid sgRNA design and off-target prediction

An offline tool available for MacOS X system is now available for free at http://www.protospacer.com/. The software combines the advantages of various CRISPR design algorithms. It features single or paired target design of gRNAs for which cleavage efficiency is estimated based on the Doenech score and off-target sites are predicted and validated. The software can handle very large genomes, custom sequences or user defined genes. gRNA design is only available for S. pyogenes Cas9 for N(20)NGG sites. It has a graphical user interface and does not require programming skills.  The workbench also connects to the IGV viewer.
Source: http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3291.html

Chemically modified guide RNAs facilitate genome editing in primary human cells 

A recent work from the Porteus lab (Stanford, California) presents the use of chemically altered gRNAs to improve low efficiency genome editing rates in primary human T cells and CD34+ hemopoietic stem cells. Modified sugar-phosphate backbone renders the gRNA more stable in cells leading to enhanced gene engineering, including more efficient non-homologous end joining and homology directed repair rates. These gRNAs have a particular advantage over unmodified gRNAs when the system is delivered as a ribonucleoprotein particle (RNP, i.e. Cas9 protein and gRNA) or as pure RNAs (Cas9 mRNA and gRNA), advantages over plasmid DNA expressed Cas9 are less clear. The authors claim there is somewhat better on/off target ratio with modified gRNAs, but we have to be cautious with this: the authors modified the terminal bases on the gRNA, perhaps functionally truncating them. Truncated gRNAs have significantly higher specificity according to the paper of from the K. Joung lab. Nevertheless, this system could be highly advantageous when working with primary cells, where genome editing rates are relatively low.

If you are interested in the paper, you can find it here.

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