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

A collection of CRISPR tools is available - includes web-based tools for guide RNA design, off-target analysis, NEHJ/HDR analysis, list of plasmids, CRISPR libraries and cloning guides. Probably I still miss a lot, let me know if your favorite resource is not included in the collection. Go tohttp://genedit.blogspot.com/p/new-page.html!

CRISPR happenings - Cold Spring Harbor Laboratory hosts the 'Genome Engineering: The CRISPR/Cas9 revolution' meeting between September 24-27, 2015. The meetings is organized by Jennifer Doudna, (University of California, Berkeley/HHMI), Maria Jasin (Memorial Sloan Kettering Cancer Center, NY) and Jonathan Weissman (UCSF/HHMI).

Oral sessions will feature CRISPR Biology, DNA Repair/Genome Editing, Human Genome Engineering, Model Organisms/Plants
Technology Development, Stem Cells/Cancer.

Abstract submission deadline: July 3, 2015. Stipends might be available for students.

More info here: http://meetings.cshl.edu/meetings.aspx?meet=crispr&year=15

Improved off-target prediction by incorporating chromatin data by the CROP-IT algorithm

Off-target cleavage is a big issue in genome editing, particularly with CRISPR. It seems that, although way simpler to use, CRISPR is not as specific as ZNFs or TALENs. A further problem is that predicting off-target sites is very challenging, for example in the recent report of Tsai S et al., an unbiased screening for off-target double strand breaks revealed sites that were not predicted by the MIT CRISPR tool or the E-CRISPR algorithm. The prediction programs however returned several potential sites that did not appear to be off-target sites. More accurate prediction of off-target sites is crucial since most researchers base their guide RNA design of these algorithms. Since for each gene, a huge number of guide RNAs could be potentially designed, accurate prediction of off-target sites would help to score gRNAs based on specificity. At this point we are very far away from this. One major reason for low efficiency prediction is differential accessibility of the DNA depending on chromatin state. The Adli lab (University of Virginia) now developed an improved algorithm for off-target search, the CROP-IT, which incorporates chromatin information.

The CROP-IT allows to search for sites up to 6 mismatches (cleavage) and up to 9 mismatches (binding). This is already an important discrimination, since certain applications (e.g. epigenome editing) requires only Cas9 binding, but not cleavage. Binding or cleavage tolerates different mismatches and now this feature is built into this prediction software. The prediction scores mismatches in relation to the PAM site and then incorporates DN-ase I hypersensitivity information into this score. The PAM site relation scoring is based on training of the algorithm on available ChiP-Seq and GUIDE-Seq data. DN-ase I sensitive sites reflect more open chromatin and more accessible DNA, however this is specific for each and every type of cell. The CROP-IT uses an average from 125 different human cell lines at this point. CROP-IT seems to outperform current prediction algorithms in Cas9 binding and cleavage prediction when validated by ChiP-Seq (Cas9 binding) and cleavage (GUIDE-Seq).

 It is available for S. pyogenes, NGG and NNG PAM sites are analyzed separately.

Link to the paper: http://nar.oxfordjournals.org/content/early/2015/06/01/nar.gkv575.full
Link to the CROP-IT tool: http://cheetah.bioch.virginia.edu/AdliLab/CROP-IT/homepage.html

New Technology based on CRISPR technology: the CRISPR-display.

The John Rinn lab (Harvard, Cambridge, US) utilizes the CRISPR system to bridge RNA cargos onto specific genomic loci enabling the targeting of different RNAs or ribonucleoprotein particles to DNA. The spectacular technology is based on using the dead (inactive) Cas9 /gRNA linked to an RNA of choice (either covalently or by hybridization). Interestingly various RNA cargo could be linked, including natural long-non coding RNAs, aptamers or pools of RNA sequences. This technology has diverse applications, i.e. analysis of lncRNA function, screening for RNA based drugs influencing specific genomic loci, imaging, modular transcriptional regulation.

If you work with lncRNA or RNPs you should check it out!

http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.3433.html

A nice figure showing the rapid evolution of CRISPR research, you can also find out the top 5 patent applicants in the technology! (source: Nature, 'The Disruptor', http://goo.gl/MYEMjR)