see latest Rudy Jaenisch Cell paper (2013) Cell 153 1-9

http://www.ncbi.nlm.nih.gov/pubmed/23643243

I was about to say the same as Francios suggested. You could also look for additional info here; http://www.genome-engineering.org/crispr/

Hope this helps



EpiGenie: Genome Editing with the CRISPR/Cas System in Zebrafish

CRISPR/Cas is getting a lot of attention lately as a way to insert or delete genomic sequences in mammalian cells, but what about in model organisms? A new paper now shows that it works well in zebrafish and suggests it could be a new “go-to” method in other models as well.

Last month, we told you about the pros and cons of CRISPR/Cas compared to two more popular genome editing techniques, zinc finger nucleases (ZFNs) and transcription-activator-like effector nucleases (TALENs). To recap, the prokaryotic CRISPR/Cas system is kind of an immune system in bacteria and archaea that researchers have adapted to make changes in mammalian genomes. It works just as well or better than the other methods and is easier to do.

Not much is known about CRISPR/Cas in model organisms, so the team from China tried it in zebrafish. Here’s what they found:

• They could make site-specific indels in the zebrafish etsrp, gata4, and gata5 genes.

• The frequency of indels was about the same as that reported for TALENs.

• The phenotypes were what you’d expect for mutations in these genes, which suggested biallelic conversion.

• They inserted mloxP precisely into the zebrafish genome, which they say shows great promise for making conditional knockouts in zebrafish, something that has been impossible until now.

“This work is one of the first two reports describing a highly efficient CRISPR system for genome editing in model organisms, which will facilitate cost-effective, genome-wide, functional analysis of coding and non-coding RNAs in vertebrate animals, including humans,” says Jing-Wei Xiong, Ph.D., who is at the Institute of Molecular Medicine at Peking University, and is one of the corresponding authors.

Find out if this could work for you at : Cell Research, April 2013.

For how long did you breed the 70% chimeras and from which mouse line were the EUCOMM ES-cells derived? I am asking because we are having really good experience with most EUCOMM ES-cell lines. If you really want to start once again, I would recommend to order the EUCOMM targeting vector and redo the gene targeting. The CRISPR technology is brand new and sexy and of course you'll find publications about positive gene targeting results. Nevertheless it is still connected to risk and failure and if you take into account all the controls that you have to do, I really would recommend to stick to the more traditional way. Why are you aiming for CRISPR anyway, why not TALENs?

Best wises

Stefan

Polygene AG

Just a quick word of caution. See Fu et al in Nat. Biotech [doi:10.1038/nbt.2623] who demonstrate very convincingly the off-target effects of CrispR-mediated genomic editing - effects rather conspicuously absent in the Cell paper [10.1016/j.cell.2013.04.025. ]. Perhaps Jaenisch and colleagues are now deep sequencing CrispR genomes to see the extent of off-target effects in their mice.

Just coming back from the Innovative Mouse Model meeting from Leiden. Convincing experiments and results have been shown that clearly demonstrate: the CRISPR technology isn't ready yet for sequence specific genome engineering purposes; too many off site effects, far more than can be tolerated and far more than observed with ZFNs or TALENs.

I work on CRISPR systems in bacteria and just came from the CRISPR focused meeting and it sounds like the technology is working very well in mice and other models, with different labs having successfully created SNPs and indels in hundreds of different genes in eukaryotic cells. The main advantage of CRISPR over TALENs and ZFNs is that it is an RNA-based system, so you simply have to clone a sequence homologous to a portion of your gene of interest (short... ~50bp or less) and co-express it with a CRISPR gene, Cas9. Cas9 will cause a double-stranded break and then you can insert sequences, cause SNPs, frameshifts, etc. I believe people are getting CRISPR plasmids from Addgene with good success.

Just an update on this discussion: two new papers published in Sep 12th Issue of 'Cell' (Rudolf Jaenisch's group and Feng Zhang's group) where they show that targeted knock-in and conditional kncckout mice (Cre-LoxP) can be generated using CRISPR/Cas9 system.

It works beautifully in mice.  No more complex design (with conventional KO and TALENS) and very fast and cheap.  About off-targets:  it's hyperbole .  In mice unless the off target is in LD with your desired mutation, it can be bred out in 1-2 generations and even still, the off targets are primarily in intergenic noncoding sequence space ad exist in a homozygous state.  George Church pub'd a beautiful paper showing wtCas9 binding at many places in genome, but only creating dsDNA breaks at very few places.  Most people (including our group) don't see much off-targeting.  Please take a look at our recent paper, the very first regulatory KO in mice using CRISPR-Cas9.  It took less than a year to make the mouse, generate the data and publish the paper.  Can someone say Nobel Prize this year or next probably to Jennifer Doudna and Emmanuelle Charpentier.

Article CRISPR-Cas9 Genome Editing of a Single Regulatory Element Ne...