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.  

The reported off-target effects in the recent Liang et al human embryo #CRISPR paper are partly incorrect.

As widely reported last week, a group in China has published results of CRISPR editing experiments in human triponuclear embryos (Liang et al, Protein & Cell 2015).   The news blurb in Nature is worth a read to get the context of the paper, which follows on the heels of a previous statement published in Science by leaders in the CRISPR field and others, in which they discourage CRISPR experiments in human embryos at this time pending further discussion of the implications of such research.    

In this post I won’t get into the ethical implications of the paper (which is more than I can deal with in one post anyway!).   Here I’ll discuss the technical results.   The Liang et al paper does not actually present much data that is very surprising - it is not unexpected that CRISPR can induce targeted mutations in humans, since it works in basically every species in which it’s been tried.    Their target gene was HBB (beta-globin) and they attempted HDR using the familiar approach of coinjecting Cas9 mRNA, guide RNA, and donor oligo ssDNA.

Here’s their 4 main points, paraphrased from the abstract:   

1. Efficiency of HDR was low.
2. Edited embryos were mosaic.   
3. Off-target mutations were evident.
4. A separate, highly homologous gene (HBD) could serve as donor template for repair, thus introducing sequences inadvertently from the other gene into the target gene.

Of these, points #1 and 2 were not surprising to those who have injected CRISPR reagents into mouse embryos, and not all that different.   The reported HDR efficiency was 14%, which is in line with at least some published mouse experiments (e.g. Singh et al 2014).   I will state that in our mouse core we are apparently seeing HDR results around 10-20% efficiency across several experiments.    Mosaicism has also been previously reported in CRISPR mice (Yen et al 2014).  Point #4 was kind of novel, but in retrospect not completely weird, since the HBD gene (delta-globin) is over 90% identical to HBB.

Ok, so regarding point #3 - off-target mutations...I was very interested in this because the authors reported four distinct off-target (OT) mutations in human embryos associated with the single CRISPR guide RNA they used, and this has already been described in the media as being substantially higher than OT rates in animal embryos.  Meaning, mouse embryos.      

One of these OTs was particularly surprising, as it looked like a very poor match to the target protospacer indeed - although the 3’-most 11 bases (the “seed” region”) matched the target, the 5’ bases only matched 1 out of 9 bases, for a total of 8 mismatches.    Frankly, this really scared me, because if true it means that the current methods used to predict OTs are not nearly broad enough.   But this level of OT mismatch was much greater than any OT I had seen before.

Bottom line: After looking at their data, I now firmly believe that only one of the four OT mutations were actually new mutations caused by CRISPR.  The other three were simply polymorphisms, already present in the  germline, that the authors mistakenly classified as OTs.

Here is how they did their OT analysis and my interpretation of the data.   Tripronuclear human embryos were obtained from a fertility clinic; you can identify these microscopically at the 1-cell zygote stage.  Fertilized by 2 sperm by accident, they are effectively triploid, and are absolutely unable to survive to term as normal pregnancies - but they can survive well enough during short-term CRISPR experiments, in which the embryos are only kept alive for a few days in vitro.   Briefly, 86 tripronuclear embryos were injected; 71 survived the injection; 56 of these were GFP-positive (used to as a reporter to show expression of injected reagents) and used for DNA analyses of on- and/or off-target effects.   28 of these embryos had on-target indel mutations and/or the desired HDR edit and were used for OT analysis.   

Note that they had originally chosen this particular CRISPR target from 3 potential targets they looked at in their gene;  one of these didn’t cut well and was not used further . For each of the other two, 7 sites were identified as the “top” potential OTs by using the MIT tool.  Of the two targets, one was found to have no OT mutations at the 7 potential OT sites when it was tested in 293T cells.  So they decided to work with this CRISPR target.  

2 of the 7 OT’s were found to have mutations in the injected embryos.  These were named G1-OT4 and G1-OT5 and are the first two of the four total OT mutations they claimed to identify (Figure 3A).  T7 mismatch assays were used for this analysis.   293T cell transfections with the guide RNA had already shown a lack of mutations across the 7 OT sites, that is, they were negative by T7 assays.  I’ll come back to these later.

They then did whole-exome sequencing on six of the embryos to identify potentially even more mutated OTs.  From this data, they first called indels and SNVs (single nucleotide variants) and then searched for protospacer similarity “allowing for ≤6 mismatches or perfect match of the last 10 nt 3′ of the gRNA” anywhere within 100 bp of the indels.  (Not sure if they did anything more with the SNVs.)   This identified two apparently new OTs, in the 3’ UTRs of  the C1QC and TTR genes, each found in one embryo (Figure 3B).  These were confirmed by T7 assays.

So - what does the T7 mismatch assay really indicate?  It reveals heterozygosity within the PCR product.   Of course, new mutations can cause this.  But so can plain old polymorphisms.   This is a drawback of using mismatch assays when applied to polymorphic samples.     

The next question is, simply, are there common human polymorphisms in the PCR products used in the OT analysis?  It’s easy to check this using the UCSC genome browser and the 1000 genomes site.   

For OT #1, a.k.a. “G1-OT4”, (Fig. 1C and 3A; PCR, hg19, chr11:132761837-132762356; intron of OPCML) there are no known common polymorphisms within the PCR that are close to the OT.    The closest SNP, rs79549129, has a minor allele frequency (MAF) of 1.2% but zero in asian populations.   There are no other annotated variants near the OT with a significant MAF.  The closest “common” SNP is rs2659601 but it’s about 50 bp from one end of the PCR product.  I don’t think that could produce the band sizes seen in the Fig. S3 T7 assays. From what I can tell from their Fig. S3 & S4, their T7 assays are compatible with new mutations that have been induced by cleavage close to the CRISPR OT site.   Thus, these look like “real” CRISPR OT effects at this site.    6/20 of on-target embryos, or 30%, had mutations at this OT.  So this looks like a real OT effect that replicates across embryos, but not in 293T cells.   

But then, polymorphisms become apparent in the other OTs...

For OT #2, a. k. a. “G1-OT5”, (Fig. 1C and 3A; PCR, hg19 chr22:31000551-31001000; intron of TULP4), there are two common SNPs on either side of the OT:  rs616358 (G/C) and rs628203 (T/C).   Haplotype derivations in Southern Han Chinese suggest haplotype population frequencies of ~56% GT, 35% GC, 9% CT, and zero % CC.  So we would expect to see plenty of heterozygosity in this PCR - easily observable by T7 assays, in the range of 50% or so being positive in a population based sample from this geographic location  - no CRISPR required.  This OT was a false positive.  

UCSC screen grab showing SNPs close to the OT (black bar in middle)

This leaves the two additional OTs they discovered by whole-exome sequencing.  Remember their workflow: they called indels in their data sets, then looked for nearby partial matches to the CRISPR target.   However, they apparently did not filter out known polymorphisms first.

OT #3 was in the 3’ UTR of the TTR gene.   Inspection of the OT sequence location (given in Fig. S6) on the UCSC browser clearly shows that rs143948820 is a known 9-base indel contained completely inside the OT.       Turns out that it’s uncommon outside of Asia but it has a MAF of ~2% in Southern Han Chinese.  With a heterozygosity of ~4% in normal diploids, it’s totally possible that 1 out of 6 triploid embryos would carry this variant.  This OT was very likely a false positive. 

UCSC screen grab; OT is black bar, indel variant is long red bar.

Finally, OT #4 was in the 3’ UTR of the C1QC gene.   And similar to the case above, this OT overlaps with a known 17-base indel, rs142916975, that has a MAF of 38% in Southern Han Chinese.   Heterozygosity should be close to 50%.   In fact, I’m surprised they got a false positive in only 1 of their 6 samples.   This is almost certainly a false positive.

UCSC screen grab; OT in black, indel variant in blue.

In summary, only 1 of the OTs holds up to scrutiny.    Importantly, neither OT found by exome sequencing holds up.  This flips their conclusion on its head: “Our whole-exome sequencing result only covered a fraction of the genome and likely underestimated the off- target effects in human 3PN zygotes.”.   While it’s certainly possible that some more OTs could be found by whole genome sequencing, the exome data was essentially totally negative.   Note that they chose a CRISPR to work with because it had a low apparent OT rate in 293T cells.   In retrospect, it was just by luck that G1-OT5 has a negative T7 assay in 293T cells.  It could have been heterozygous, but it's apparently not.

To their credit, the authors rightly restate that it’s going to be critical moving forward to carefully analyze off-target effects in any human applications of CRISPR.  This is widely agreed upon (see below).  However this paper made some technical mistakes in this regard.  While underestimating off-target effects could certainly have serious negative consequences for future CRISPR-based clinical treatments for genetic disease - and nobody wants that - overestimating them could generate an excess of hesitation to research the feasibility of such treatments within the broader scientific community.  

As stated by Baltimore et al in their Science commentary:

“It is critical to implement appropriate and standardized benchmarking methods to determine the frequency of off-target effects and to assess the physiology of cells and tissues that have undergone genome editing.”

I don’t think I’m blowing smoke here that we all need to get this right, as the media quickly reported on the Liang et al conclusions:  

From Wired:  “But—and this is a big but—using the technique without proper guidance could result in unforeseen consequences. The Chinese researchers, for example, found mutations in many of the embryos in genes other than the ones they’d targeted with CRISPR/Cas9.”

From Time, quoting Carl Zimmer from National Geographic:   “The experiment “came out poorly,” Zimmer says; in some cases, DNA was placed in the wrong spot and “off-target” mutations were discovered in the DNA.”

From the Washington Post:   “And in some of the embryos, the gene editing caused unintended mutations in other genes.”

From USA today (emphasis is mine): “The team also found that the complex used in the procedure was also acting on other parts of the genome, leading to other bits of it mutating. That happened much more than in previous experiments on adult human cells and animal embryos — and could happen yet more if the whole genome were used, as it would be if the embryo were to be implanted.”

            (OK, note from this last article the specific comparison to the very observations that I have blogged about in more detail than most people probably ever wanted to hear about...My point is that, due to the technical problems in the Liang paper, I don’t think we can yet say the off-target effects were “much more than in previous experiments on adult human cells and animal embryos”. )

One final note - my analysis of this paper should not be interpreted to mean that I fully endorse CRISPR experimentation or applications in human embryos.   I also applaud the authors' cautionary tone that the incomplete efficiency of CRISPR editing in humans is a problem that any therapeutic applications need to address.

Whew, this was the longest post yet.   

Are there sequence preferences near the 3' end of the #CRISPR protospacer? Paper from the C. elegans field explores this.

When the first word in a paper title is "Dramatic", I certainly wonder if I will agree after reading it…It's worth a blog post at any rate.   This paper by Farboud, B. and Meyer, B.J. is titled "Dramatic Enhancement of Genome Editing byCRISPR/Cas9 Through Improved Guide RNA Design" (Genetics, Vol. 199, 959–971 April 2015).   

As is true for other model organisms, CRISPR is very useful in nematodes for performing mutagenesis.  In this paper the authors were inspired by the previous observation that the Cas9 protein physically associates with the PAM motif (NGG) sort of promiscuously across DNA.  This had also previously led to the discovery that - in vitro - a CRISPR target region that is generally rich in GG dinucleotides will enable higher rates of cleavage at a unique CRISPR target within that region, than if the region is otherwise reduced in GG content.  In other words, general GG density probably "attracts" Cas9 and keeps more of it around, which in turn may enable faster recognition of the actual target.

Using this idea, the authors tested whether simply keeping an extra GG motif nearby the actual PAM NGG motif would enhance CRISPR mutagenesis in worms.  Turns out that if the PAM is followed (3') by another NGG, it doesn't help.  However, if the first 3 bases 5' to the PAM are NGG - that is, the last 2 bases of the protospacer are GG - they saw a consistent, and yes, dramatic improvement in recovery rates of CRISPR mutants.  This was a pretty striking finding and was validated across about 8 to 10 sites.  For comparison they tested "shifted" targets where they just shifted the protospacer 5' by 3 bases, and used those last 3 bases of the first protospacer as the PAM as the control.   These were almost uniformally poor in terms of absolute numbers of mutagenesis, with numbers usually at zero - meaning with their particular system of worm injections the baseline rate is pretty low.   The targets that had the GG at the protospacer 3' end usually had high mutation rates in double digit percentages.

So is this observed in other animals/cells/systems?   Well, based on my own work and from what I see in the literature, in mice we definitely don't need to have a GG at the protospacer 3' end to get high efficiency mutagenesis.   There are still no consistent rules here, but there are trends for sure.  Cas9 does seem to prefer purines in the last two bases of the protospacer as this seems to enhance gRNA loading (Wang et al 2014).     GC richness across the protospacer is definitely good (Gagnon et al 2014) which must correlate with G's in the protospacer 3' end.  Interestingly, Doench et al 2014 observed a preference for purine in the last base of the protospacer, but not much preference for the penultimate base (see their Fig 3a).

On the other hand I can't identify many examples yet of mammalian CRISPR targets that were published, had a GG in the last bases of the protospacer, had hard mutagenesis rates published and enough other targets in the same paper for a good comparison.