#CRISPR target sequence preferences are being clarified. Xu et al Genome Research paper.

It's of huge value to be able to predict CRISPR target efficiency ahead of time.  Xu et al have published an analysis of multiple guide RNA data sets and extracted what they claim is an improved model for target cleavage efficiency prediction.   This data is all for the S.pyogenes native Cas9. 

Xu et al. Sequence determinants of improved CRISPR sgRNA design.
Genome Res. 2015 Aug;25(8):1147-57. doi: 10.1101/gr.191452.115. Epub 2015 Jun 10.

Their paper is important to me for several reasons.  First, they have examined two independently-published "large" guide RNA data sets that had mutagenesis-efficiency data, which allows more confidence that trends of sequence preferences are holding up across labs and platforms.   Second, they validated their predictive model on a small (in comparison to genome-wide, but still not bad) data set of new CRISPR targets and corresponding guide RNAs.  Third, they did "in silico validation" by turning their model loose on another target/indel data set, and showed improved performance of their predictive model over a previously published model.   See ROC curves in Fig. 4b.    This allows an ability to weed out "50-60% of the inefficient sgRNAs…at the cost of 10-20% of efficient sgRNAs misclassified."  That is, misclassified as inefficient.    

For those who are interested in genome-wide knockout screening experiments these sorts of models are very good for increasing efficiency of the screens.   Moreover, if you wish to knockout particular genes, it will allow you to test or use fewer targets per gene till you find one that works well.

OK, now the sobering reality for nerds like me is that predictive models, even with great ROC curves, have false positive and false negative rates that will bite you in the behind on a regular basis if you are designing large projects around the function of single CRISPR targets.  I'm still facing this issue for precision knock-in projects, for which there are often not many targets to choose from.   And with transgenic mice we always want the efficiency as high as possible.   For cell lines, hey, that's not as much a problem if you can subclone the edited lines.

But let's get back to the CRISPR target sequence preferences.  The bottom line here is that the last three bases of the protospacer seem to have the most influence on cleavage efficiency, with a C preferred at the -3 position (relative to the PAM), and G's at -2 and -1.    Also, G's are helpful at the -17 to -14 region, while A's are good at the -12 to -9 region.  Finally, a C seems helpful at +1 following the PAM.

Looking back at the Wang et al paper, they also reported a preference for A's at around -10 to -8, and essentially a "GCRR" preference for bases -4 to -1.  This makes sense since Xu have based their model partly on the the Wang data.   However, Xu et al point out that the apparent G preference at the -20 position is probably an artifact of the Wang sgRNA library in that these may have had increased efficiency due to enhanced transcription, not activity per se.

General GC-richness in the protospacer is known to correlate with CRISPR mutagenesis.  Could that just be driven by the GC-rich preferences of the last few bases?   Otherwise, GC-richness doesn't clearly emerge from the Xu model, at least to me anyway.  I took a crack at this by looking at a data set from Gagnon et al, mostly because I could handle the size of their sgRNA list in an excel spreadsheet without exploding my own brain or my iMac.  My impression is that GC richness is still "good" even when the last 4 bases of the protospacer are similar.   Here's an example.  From Gagnon et al's list of 122 sgRNAs with indel numbers, I ranked them according to how well they matched the "GCRR" of the last four bases.  I based this on the Wang et al paper although I think it is very similar to that corresponding part of the Xu model.   My  "score" ranged from 0 to 7.  Then I examined the subset of 30 targets that all had a same "score" of 5.   So these targets are all controlled, at least kinda sorta, for their  variation in bases -4 to -1 in that they have similar strength of matching to the "GCRR" motif.  Finally, I graphed the indel frequencies versus the GC content of the first 16 bases of their protospacers.   Here is the data.  y axis= indel frequency (in a zebrafish model), x axis= # of GC base pairs in first 16 bases.

This ain't close to something I'd submit for peer review but I do see a trend.  GC richness in the first 16 bases of the protospacer correlates with cleavage efficiency, even within a group of targets for which the 3' ends are similar.   So for now - I will continue to prefer overall GC-rich targets that also have at least some matching to the "CGG", or "GCRR", motif at the very 3' end.  

Also, "CGG" matches the high-efficiency 3' end reported by Farboud and Meyer so there's another corroboration.

So, the answer to my previous post "Are there sequence preferences near the 3' end of the #CRISPR protospacer? …" is, yes.  And this holds up for S.pyogenes Cas9 when used across human, mouse, fish and C.elegans models.   

A final note - these data all refer to cleavage and/or knockout efficiencies.  CRISPRi and CRISPRa screens, which do not lead to or require DNA cleavage, have different sequence preferences which Xu et al also modeled in detail.   

Happy CRISPRing.

My review of recent Joung lab paper with new PAM specificities! for Cas9: will broaden choice of #CRISPR targets.

It was just a matter of time before someone mutagenized Cas9 to try to change its PAM preferences.  (What's a PAM? Look here if you aren't hip yet).   Although that "NGG" motif is pretty abundant in genomes - heck, it's only 2 bases - it sure would be nice to be able to target even more sequences with high specificity.  For example, the closer the CRISPR site is to the site where precise genome editing is required, the more efficient it will (probably) be;  having more PAM choices will only be helpful in this situation.  But, having more targets isn't very useful unless the properties of CRISPR specificity and sensitivity remain robust.  

So now the Joung lab has led the way with efforts to coax Cas9 into preferring new PAMs, as described in Kleinstiver et al's new paper in Nature.    I really like this paper.   It introduces new Cas9 variants with preferences for NGA, or NGCG PAMs. The new variants are not complicated to engineer, maintain high cleavage activity, work in vivo, and have low off-target effects similar to native Cas9.  

Previously, Anders et al had published a Nature paper describing Cas9-DNA structural interaction.  (Senior author of this paper was Martin Jinek, who was first author of the seminal 2012 Dounda/Charpentier Science paper).  An interesting nugget in that paper was a first attempt to alter Cas9 specificity by mutating the two amino acids (Arg1333 and Arg 1335), which apparently interact directly with the two guanine bases of the PAM motif, to glutamine.  This was inspired by Cas9 variants from non-S.pyogenes species which prefer A-rich PAMs and have glutamines in the homologous positions.   However these changes alone couldn't make S.pyogenes' Cas9 prefer NAA instead of NGG.

Enter Kleinstiver et al.  They began a systematic attempt to engineer new PAM specificities into S.pyogenes Cas9.  First, they used a clever bacterial assay to measure PAM preference in which the CRISPR target is within a toxic gene.  Cas9 variants with different codon changes were then introduced.  In this setup, target cleavage disrupts the gene and allows the bugs to grow, allowing one to sequence the survivors to figure out which Cas9 variants worked.  In this manner they identified combinations of codon changes that allowed Cas9 to recognize a NGA PAM.  2 variant combos, "VQR" and "EQR" , emerged as being best at now preferring NGA over NGG.

Then, they used a different assay to measure preference for all the possible different PAMs for selected Cas9 mutants.  See Figs. 1e, 1f for these data nicely visualized.  For example you can clearly see how wild type Cas9 greatly prefers NGG, but has a little ability to use NAG as had been previously reported by many - in fact many off-target analyses consider NAG PAMs as well as NGGs.   Then, they tested the VQR and EQR variants, which revealed that these now are sensitive to the fourth base in the PAM.  Specifically, Cas9-VQR "likes" NGAG, NGAA, NGAT, NGCG the best.    Interestingly, Cas9-EQR preferred NGAG almost exclusively.  The authors concluded that the T1337R variant is a gain-of-function allowing sensitivity to the fourth base, which is then specified by other codon variants.  Cool.

Next, they found that the "VRER" combination allowed specific preference for a NGCG PAM.  Note that this GCG motif is much less common in mammalian DNA than the other PAMs - after all, it's got a CG in it - but that also means it's off-target potential is lower.   Since I know lots of genes with GC-rich regions I'm betting this PAM will come in handy.

A few more points from the paper:   The new variants work in zebrafish in human cells, and have good activity and low off-target effects.  Additionally, they noticed that the D1135E variant actually increased PAM specificity for the wild-type NGG PAM relative to NAG - see Fig 3a, and furthermore it reduced off-target cleavage on other off-target sites that have mismatches in protospacer but (presumably) the NGG PAM.  In other words D1135E reduces off-target cleavage in general (at least somewhat) without reducing on-target cleavage.   Sounds good to me!

Finally, they examined two Cas9 genes from other bacterial species and showed they could carefully measure their normal spectra of PAM preferences (which are different from the NGG of S.pyogenes).  In other words they are poised to do the same mutagenesis work on these other Cas9 proteins, which will add even more PAM choices to the toolkit.   

i was about to write "these Cas9 variants should be widely available soon", then I thought Hmm, better check Addgene.   Sure enough:   VQR, EQR, and VRER expression plasmids are already available!  Kudos to Keith Joung and his lab for making these available to the world.  Happy CRISPRing with new PAMs!

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.

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!

@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.
http://www.ncbi.nlm.nih.gov/pubmed/25897126

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 http://www.nature.com/nmeth/journal/v12/n6/full/nmeth.3408.htmlhttp://www.ncbi.nlm.nih.gov/pubmed/26020497

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!

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

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 

33, 

538–542

 

(2015)

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.

About using DNA or RNA for mouse embryo #CRISPR injections.

I got a question:

Isn't the disadvantage of injecting DNA the threat of integration and more frequent mosaicism than in the case of RNA as Cas is expressed quicker? Do you have some direct experience with that? Thanks! 

Um, well yes.  Yes.  Those are the disadvantages.  Also I will add that because the RNA should lead to quicker Cas9 expression,  mutagenesis efficiencies will likely be higher than with DNA vectors.

So why use DNA at all?  Well, the issues are mostly practical.  DNA vectors are easy to customize for CRISPR.  Although the issues of efficiency and mosaicism are potentially problematic, I have seen pretty consistent success* in generating simple indel mutations following injections of PX330-style CRISPR-Cas9 DNA plasmids.  That is, consistent double digit percentages of founders carrying mutations as assayed by PCR and/or sequencing.    In addition, in our core we have obtained HDR-mediated codon editing rates in the 10-20% range using PX330 vectors co-injected with appropriate "donor" oligos.  But this is dependent on cooperative CRISPR sites that have a high rate of baseline cleavage.

Another practical consideration is that not everyone can routinely synthesize high-quality RNAs in vitro with consistency.   Quality DNA is relatively easy to prepare and QC.   RNA is much less so - especially for the 4+ kilobase Cas9 mRNA.   OK, so some of you are saying "Come one, my lab makes RNAs all the time - no prob! " .    That's awesome, but the empirical observation is that it's not trivial to get proficient at making long mRNAs, and to keep on top of the key reagent issues (RNAses, enzymes going bad, etc.).

Also, CRISPR DNA plasmids are immediately useful for cell culture gene editing experiments.  Some labs will be making these anyway so they will have them on hand, ready to go.   

What I am also observing - which many others have reported - is that a fraction of CRISPR sites just don't cut very well, even when the sequence characteristics of the site seem OK.  (Like, somewhere on the order of 1/3 to 1/4 of CRISPR target sites?) Most of our injections to date have been using DNA plasmids.   It's possible that RNAs might save the day for some of these sites.    

The ability to do precise HDR-mediated editing/insertions, rather than simple indels, is very compelling and is the direction most of our CRISPR ideas are going in terms of new mouse models.  But coding modifications usually have extremely narrow CRISPR target choices that are imposed by the science;  if you want to change a codon, you'll probably need a target as close as possible - preferably overlapping the codon.  There won't be many to choose from.  So getting the highest efficiency cleavage rates may be critical for some of these projects - for these, Cas9 mRNA or protein may be needed.

Finally, these issues of target efficiency really call for pre-validation of sites.  This can be done by transfecting CRISPR plasmids into cooperative cell lines, e.g. NIH3T3 for mouse targets, followed by PCR and mismatch cleavage assays, which can then be quantified.  But then - if you go through the trouble to do that, you will have generated the DNA plasmids and thus have the DNA reagent ready for injection.   

Having said all that, although I really like the convenience of plasmids, the RNA problems are all about sourcing them.  A few vendors, such as Sigma-Aldrich can provide custom guide RNAs and Cas9 mRNA that work.  (FYI I do not receive any compensation from Sigma).  The RNA reagent expense is less than the cost of mouse embryo injections.  I suppose zebrafish researchers may balk at the cost, as they will have more capacity to inject fish eggs, in their own labs usually, and may be more willing to make RNAs in-house.  For mice, you'll be usually working with a transgenic core and spending thousands of bucks per experiment.  Vendor-supplied RNAs may be worth the money.    Thanks for the question!

*Actually, "consistent" may be misleading… To clarify, about 75% of the NHEJ projects I've been observing have had this level of success.   So - more success than not, but then again, not perfectly consistent.