Thursday, June 25, 2015

Photoactivatable #CRISPR-Cas9 systems!

There are a few recently developed light-activated CRISPR-Cas9 tools that have been reported lately.  I'm motivated to post this based on the most recent one, which demonstrated gene editing using modified "split" Cas9 protein halves that were conjugated to newly developed light-inducible dimerization domains named "Magnets".   This was a paper just published by Nihongaki et al. in Nature Biotechnology.   (PDF is as of the post only published online at this link). This system is nice in that it just requires expression of normal gRNA plus the two modified coding portions of Cas9, plus, blue light to activate dimerization and Cas9 targeting function and cleavage.   It's reversible too - removing the light stimulation lets the complex fall apart.  Neat!

Other groups in parallel have created very similar tools that allow light-inducible activation of Cas9 to allow targeting.  In a related paper Nihongaki and colleagues showed they could use this to activate transcription at CRISPR target genes using light, and Polstein and Gersbach have made very similar tools.  Both groups used the CRY2 and CIB1 light-induced dimerization domains from Arabidopsis.

Using a different strategy, Hemphill et al used a caged amino acid strategy to encode a light-activatable codon into Cas9.  This system is a bit more complex to set up, as it requires engineering a pyrrolysl tRNA synthetase into the cells being targeted - basically, re-engineering the genetic code to get a light-activated lysine into the guts of Cas9.  This seems very different mechanistically than the dimerization approach and so it maybe a good alternative for some applications, as it probably has some distinct wavelength and kinetic properties.  Always a good thing to have different tools in the toolkit.

Tuesday, June 16, 2015

2015 Gruber Prize in Genetics awarded to Charpentier and Doudna for #CRISPR.

The 2015 Gruber Prize in Genetics is being awarded to Emmanuelle Charpentier and Jennifer Doudna for their pioneering work on CRISPR biology.   This prize is presented annually at the American Society in Human Genetics annual meeting, which will be held in Baltimore this year in October.

If you're not familiar with the Gruber Prizes, they are awarded in several disciplines including genetics and they include a $500,000 cash prize, so it's quite an award. Congratulations once again to Drs. Charpentier and Doudna!

Tuesday, June 9, 2015

@LluisMontoliu guest post! about low off-target #CRISPR rates in embryos.

Lluis Montoliu is very well known to the transgenic mouse community and and expert on all things related to mouse genetic engineering.  Therefore I was very happy when he sent a message to the ISTT mailing list describing the recent in-depth confirmation that yes, CRISPR can have extremely low off-target cleavage rates in mouse zygotes, as alluded to in one of my previous posts (and probably true for human embryos too despite a recent report).   

He has kindly agreed to let me re-post his message on this blog.  Thanks Lluis!  You can also follow @LluisMontoliu on Twitter, and check out his own CRISPR information web site and also his lab's web page.  

Subject: [ISTT_list] Off-target mutations are rare in CRISPR-Cas9-edited animals

Dear colleagues,

Anyone who has already carefully analyzed mice edited by CRISPR-Cas9 will have confirmed the almost absence of off-target mutations, in contrast to what was initially predicted and announced. Off-target mutations appear to be very rare in genome-edited animals, if present at all. We and other have usually taken a shortcut and have opted to analyze a limited number of off-target sites in our genome-edited mice, selecting a few off-target sites (those with higher score, higher probability to be modified) and cloned and sequenced these DNA pieces from all founder animals generated, just to find that none of them appear to be modified.

Now, Bill Skarnes and collaborators (Sanger Inst., Hinxton, UK) have done the proper experiment, the experiment we and other would have liked to do, namely: whole deep genome sequencing on CRISPR-Cas9-edited mice. And they found the same result. Even if you don't select for sites and you review the entire genome there appear to be no off-target sites that are modified by the CRISPR-Cas9 reagents.
Off-target mutations are rare in Cas9-modified mice Vivek Iyer, Bin Shen, Wensheng Zhang, Alex Hodgkins, Thomas Keane, Xingxu Huang & William C Skarnes Nature Methods 12, 479 (2015) doi:10.1038/nmeth.3408

Hence, these amazing tools are far more precise and accurate than initially considered, particularly when these are injected as RNA (orprotein) into zygotes (into fertilized oocytes). Of course, this does not mean that you should not aim to obtain and analyze at least two independent mutant/edited animals to confirm the robustness of the associated phenotype, as you would be doing with any other genome alteration you would be producing.  And, bear in mind, the whole picture might be different in cells, particularly if they are transfected with DNA plasmids transcribing Cas9 constantly and in high amounts,  and hence providing lots of opportunities (and time) for this endonuclease to cut elsewhere, other than the expected targeted sequence. In contrast to what happens in zygotes, where a limited amount of Cas9 RNA (or protein) is used, does the job and vanishes away.

Further enjoy your genome-edited animals!

Dr. Lluis Montoliu
Investigador Cientifico - Research Scientist CSIC Centro Nacional de Biotecnologia (CNB-CSIC) Campus de Cantoblanco C/ Darwin, 3
28049 Madrid (Spain)

Friday, June 5, 2015

More confirmation that SCR7 increases #CRISPR insertion rates by inhibiting NHEJ.

I'm kicking myself for not finding this paper two months ago when it came out - I've been waiting for this sort of data!   Maruyama et al have published a more complete description of SCR7 tests in CRISPR modifications.   

Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.  
  • Takeshi Maruyama
  • ,
  • Stephanie K Dougan,
  • Matthias C Truttmann,
  • Angelina M Bilate,
  • Jessica R Ingram
  • Hidde L Ploegh.  
  • Nature Biotechnology 

    They confirm what Singh et al previously reported in a small but exciting data morsel last fall, which is that substantially higher rates of HDR-mediated insertion can be achieved in mouse zygotes by treating them with the NHEJ inhibitor, SCR7, during the injection process.   They actually mixed SCR7 (final conc. 1mM) directly into the injection cocktail of gRNA + Cas9mRNA + donor ssDNA oligo.

    After some preliminary tests in cell lines, they moved to zygotes.  Using a donor oligo to insert a short peptide tag and validated CRISPR targets/gRNAs, they did tests with and without SCR7.    Bottom line:  HDR-mediated insertion rates increased by several fold for the two genes they tested.   Although that may not sound like a breakthrough to some of you, many of you will know that in the world of mouse engineering it's key, because it will probably often mean the difference between getting zero versus a few correctly engineered pups out of an injection series.  

    Some other highlights are:

    1.  The embryos seem to tolerate SCR7 application under these conditions with no problem; no toxicity or increased death was noted.  Various other studies seem to support that transient inhibition of NHEJ is well tolerated.   Note that the SCR7 target, ligase IV, is critical for embryonic development so it can't be globally knocked out.  

    2. No increase in off-target effects.   Cool.

    Technical notes:

    1.  Yesterday's google searching quickly turned up 3 companies selling SCR7.  Yay.

    2.   SCR7 must be dissolved in DMSO.  I think making a stock solution of 100 mM SCR7 in DMSO is reasonable.  So the final injection mix, with 100-fold SCR7 dilution from the stock, will have 1 mM DMSO and also 1% DMSO.   I couldn't dig out the SCR7 stock solution details from the paper but it's probably close to these parameters.

    3. SCR7 very strongly inhibits the recovery of NHEJ-style mutations from the CRISPR targets.  

    4.  The zygote injections were all done cytoplasmic, not pronuclear, although they were done at the pronculear stage.  Thus it is clear that HDR edits with ssDNA oligos can be efficiently done by cytoplasmic injections.   This is great because it results in higher rates of pup survival than pronuclear injection.

    Still lingering questions for me:

    1. Although the authors showed they could increase the insertion rate of a "large" cassette - a GFP-style reporter ORF - in cell culture, they did not repeat this experiment in embryos.  Or at least they didn't show the data.  Was there negative data to report?   Or just not enough live pups yet for them to feel comfortable with publishing a negative result?  Or have they not tried it yet?   The routine insertion of kilobase-sized cassettes in embryos is now my next CRISPR mountain to climb!

    2.  I would kinda like to know if there may be an increased rate, or change, in the genome-wide mutation rate by SCR7 treatment. After all we are mucking around with the DNA repair pathway here.  Since each mammal embryo probably has on the order of 50-100 new mutations anyway, it would have to be a pretty substantial change in mutation rate to scare me off.   I'll bet there is no detectable effect.  Besides, NHEJ usually results in new mutations anyway - so I would imagine that we'd observe cell or embryo death following SCR7 treatment, long before we could observe a change in mutation rates or spectrum in surviving embryos.