New #CRISPR paper with more evidence that inhibiting NHEJ increases homology-mediated DNA repair. Improvements over SCR7.

Chu et al. have recently reported in Nature Biotechnology a detailed description of inhibiting the NHEJ pathway to enhance HDR (homology-directed repair) in cell culture CRISPR editing experiments.  This follows on the observation by Singh et al that SCR7, an inhibitor of ligase IV, accomplishes this in mouse embryos although the data set in the Singh paper was limited.  This new paper is very detailed in its implementation of a traffic-light reporter system to quantify NHEJ and HDR under various conditions.   

Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.  Van Trung Chu, Timm Weber, Benedikt Wefers,Wolfgang Wurst, Sandrine Sander, Klaus Rajewsky & Ralf Kühn. NatBiotechnol. 2015 Mar 24. doi: 10.1038/nbt.3198. [Epub ahead of print]

Since there's more than one way to potentially inhibit NHEJ, the authors used several approaches.  First, shRNA was used to silence ligase IV and/or KU70 or KU80.    The latter two proteins make up the KU heterodimer, required for NHEJ.     Second, they used SCR7.  Third, they coexpressed the adenovirus proteins E1B55K and E4orf6.   These are known target ligase IV for degradation. 

Before getting into more details, the bottom line was that each of these approaches (in human HEK293 cells) could both inhibit NHEJ and increase the rate of HDR at the same time.  However the approaches did this to varying degrees.  There was a clear trend that the more NHEJ inhibition occurred, the greater was the increase in HDR efficiency.   The most effective treatment was the coexpression of E1B55K + E4orf6.   Not only did this reduce NHEJ more than the other methods but it showed the greatest HDR increase as well.  

Figs. 1c, 2h, and 2j respectively suggest ~7x, ~8x, and ~3.4-51x increases in HDR were observed with E1B55K + E4orf6 coexpression as compared to the identical CRISPR/Cas experiments without these factors.

Therefore, transient NHEJ inhibition is looking better and better as a way to enhance HDR in CRISPR applications.  This will be likely be very important for gene therapy approaches, as well as just plain very useful for any HDR applications including of course, mouse embryo injections.   In fact, Chu et al go on to show that by combining flow-sorting of transfected cells with transient selection with an inserted antibiotic gene they were able to obtain essentially HDR-edited cell clones at essentially a 100% rate (e.g. supplemental fig. 13).  

Suddenly I am very interested in these adenoviral E1B55K and E4orf6 genes… These are apparently derived from the adenovirus C, a.ka. adenovirus 5, and Origene sells cDNAs and antibodies for these.    E4orf6 is also called E4orf6/7.  

#CRISPR MultiTargeter, new tool to discriminate targets in similar sequences.

This web tool and associated paper by Prykhozhij et al was interesting as it may be very useful for certain target identification situations, particularly for highly similar gene paralogs and internally duplicated exons within genes.   

If you'd like to target a conserved domain in a gene, you'll probably want to make sure the CRISPR guide RNA won't recognize the DNA encoding the same domain in other conserved members of the gene family.  Conversely, there might even be special situations where you would want to zap more than one target at the same time by actively choosing to use a shared CRISPR target that's present across conserved genes.   As the title implies, this web tool can find the shared and unshared CRISPR targets among a group of similar sequences.     

I've also thought about this idea for picking targets in which a SNP would disrupt guide RNA binding.   For example, in a situation where you'd like to bias in favor of specifically only modifying one of the two alleles in a cell, you could use a SNP-specific CRISPR to target a heterozygous embryo, cell line, or whatever, to increase the odds that only one of the two alleles is modified.    (It's not a perfect idea as single base changes don't always change CRISPR target specificity - though they seem to usually do this if they are in the "seed" region.)   This effect might be good in situations where you really want to generate a mutant allele, but the homozygous mutant is absolutely lethal.     Mouse pronuclear injections are often done on hybrid strain backgrounds so there's lots of heterozygosity to be exploited for this.  If it's useful.  Maybe??

PLoS One. 2015 Mar 5;10(3):e0119372. doi: 10.1371/journal.pone.0119372. eCollection 2015.CRISPR MultiTargeter: A Web Tool to Find Common and Unique CRISPR Single Guide RNA Targets in a Set of Similar Sequences.Prykhozhij SV, Rajan V, Gaston D, Berman JN.

Check out Kelly Rae Chi's article in The Scientist about new #CRISPR innovations. tinyurl.com/p7ljrmt

This new article in The Scientist discusses these 4 new CRISPR innovations, two of which I've also blogged about before  (but check out the article for more comments from the authors…)

(1) using SCR7 to promote homology-directed repair (HDR) in mouse embryos; 

(2) engineering cells to contain an inducible Cas9 gene; 

(3) improved methods for directly delivering Cas9 protein into cells; and 

(4) more thorough off-target identification using COSMID.   


All interesting and worth reading about!   

#CRISPR donor DNAs can inhibit targeting if you don't disrupt the target in the donor. Parikh et al PLOS One paper.

This is a new paper that is nice as it presents a lot of detailed results across a series of test CRISPR injections in mice. 

Detailed Phenotypic and Molecular Analyses of Genetically Modified Mice Generated by CRISPR-Cas9-Mediated Editing.  Bijal A. Parikh, Diana L.Beckman, Swapneel J. Patel, J. Michael White, Wayne M. Yokoyama.  PLOS ONE |DOI:10.1371/journal.pone.0116484  January14, 2015 

It's always tough to generate lots of numbers across mouse injections.  It's just a lot of work and expensive.  Despite some small numbers in the individual experiments, and some repeat description of indel mutations, etc. that others have reported, this group presents some new insights that are good to know.  The most important one I got from this paper was an interesting observation concerning their gene repair experiments.   

The target gene in this case was Tyr, or Tyrosinase, which of course when completely inactivated causes albinism.  This group did several good comparisons of different targets, nickase vs. "native" Cas9, and also injections on C57BL6 ("B6")versus albino C57BL6 ("AB6").  Thus, they could make induce mutations disrupting wild type Tyr in the B6 strain - generating albino mice - while attempting to repair the already-mutated Tyr in the albino AB6 strain.  

To do the latter they tried using as the donor a wild-type ds-DNA fragment with 800 bp homology arms flanking the wild type region of the CRISPR target(s).   This failed to repair Tyr in AB6 mice, which in of itself was not a tragedy - CRISPR-mediated repair is not as efficient as simple mutagenesis and the sample size was low.  But what they also observed is that these injections generated zero indel mutations, even though they showed the same CRISPR reagents generated lots of mutations at the target sites just fine when they were injected WITHOUT the wild type donor DNA molecule.  Therefore they repeated the experiments with a modified donor DNA fragment that had sense mutations disrupting the CRISPR target site.  This worked well in both generating a few correctly repaired alleles, but also restoring high rates of indels generated by NHEJ, thus proving that the wild-type molecule itself was inhibiting the CRISPR machinery.  Since the donor was a double-stranded DNA, it makes sense that it was "soaking up" the Cas9/sgRNA,  It was certainly present at large stoichiometric excess to the chromosomal targets.   

I counted up the gene repair injections they did with either the WT or modified donor DNA fragment, and the summary was as follows:

Experiment :     (Mice with indels/ Mice with HDR-mediated repair / Total number mice)

Wild-type donor DNA:   0 / 0 / 27.  Across 3 experiments,
Modified donor DNA:  18 / 3 / 41.  Across 5 experiments.

These experiments were a mix of nickase vs. native Cas9, 2 different targets, and B6 and AB6 mice.  But the trend seems like it's clear - CRISPR-mediated mutations were strongly inhibited with a wild-type donor DNA that carried a perfect match to the CRISPR target.  

Would a single-stranded donor oligo with a wild-type target match also inhibit?  It's not clear to me, as I think all the oligo edits I've seen published disrupted the target so that doesn't provide data;  the single stranded DNA probably won't complex with Cas9.

TIDE: an online tool for evaluating #CRISPR gene editing in sequence trace files.

TIDE is a neat new web tool that's designed for a specific problem that I've definitely been dealing with. Following a CRISPR experiment, either in cell lines or animals, it's not trivial to quantify how well editing/mutagenesis worked and what sort of mutations were generated.   This is well summarized in the introduction of this paper so I won't repeat that, but I have certainly had these situations:  first, staring at ABI chromatograms following sequencing of PCR products from founder mice, and second, trying to quantify cleavage in pools of transfected cells.     Of course, the target site PCRs are going to usually contain mixtures of molecules with different mutations, and likely some amount of wild-type allele (for sure in pooled cells, often in founder animals).   So direct sequencing is hard to interpret as the actual chromatogram data past the cleavage site is usually a jumble of overlapping staggered sequences.

What TIDE does is actually to quantify the underlying non-wild-type sequence signal in the chromatogram data 3' to the expected cleavage site, then it quantifies the apparent contribution of specific, underlying mutant alleles, based on the pretty good assumption that most of the mutations generated by CRISPR will be short indels.  This seems to be a extension of PolyPeakParser, which I blogged about previously, but it's able to deal with multiple mutant alleles.  

I had a recent data set of sequence files from a mouse CRISPR experiment, so I thought I'd compare our independent analysis of the founder mice to TIDE's interpretation.  The gene is anonymized but I can state that it was a straightforward attempt to create indel mutations in a gene of interest.    Here's what we did:   About 25% of pups were positive for new mutations as revealed by Surveyor assays.  We then sequenced PCR products on 8 founder littermates, of which 6 were known Surveyor-positive and 2 were of unknown status.    

The last 2 (#19, #20) mice had normal, wild-type sequencing data.   The other 6 mice had very jumbled sequences past the cleavage site.   After some serious staring at the chromatograms - which took a while - I made some guesses that some of them had specific indel mutations.  However some of them were just too complex for me to figure out.      

Then I analyzed all of them with TIDE, using the sequence file from wild-type mouse #20 as the control file (which TIDE requires).   Here's the results:

Pup #	Pre-TIDE manual interpretation	TIDE result
2	WT allele and at least 2 different mutant alleles present. Could not interpret mutations at all.	No significant results, but the sequence quality was rather poor to begin with.
6	WT allele and a 1-bp deletion allele. Germline transmission confirmed.	66.5% WT, 24.8 % 1-bp deletion.
10	No WT allele; one 3-bp deletion; plus a complex (discontiguous)  4-bp deletion.  Germline transmission confirmed of both alleles at essentially mendelian rates.	10.9% WT, 44.9% 3-bp deletion, 33.7% 4-bp deletion.
16	WT allele and 2 different mutant alleles present. Could not interpret mutations.	22.5% WT, 58.5 % 1-bp deletion, 9.7% 5-bp insertion.
22	WT allele and a 1-bp deletion allele.  Germline transmission confirmed.	60% WT, 30.2% 1-bp deletion.
24	No WT allele, but multiple (>3) mutant alleles.	At least 4 different deletions of -2, -12, -28, -29 bp, each at low levels.
19	WT allele predominates.	75% WT;  7.4% 2-bp insertion; 8% 8-bp deletion.
20	WT allele predominates.	(Used #19 as control) 82.8% WT, 10.7% 4-bp deletion.

I was fairly impressed by the TIDE results.  First, it agreed with my specific interpretations for #6, 10 and 22, which were actually confirmed by germline transmission.   Second, it was able to correctly call 2 mutations at the same time in mouse #10.   Third, it made interpretations that made sense for founders #16 and 24, which I had given up on.    

Finally, I didn't really give the algorithm the optimal control sequence.  Instead I used the file for an apparently wild-type founder animal (#20).  However - when the files from #19 and #20 were used as controls to analyze each other, low levels of mutant alleles were detected.  And yes, if you go back to the chromatograms you can see a little underlying signal that may be a bit more than "usual" past the cleavage site - but it's very easy to miss.   This result is actually consistent with the experiment, since the embryos were injected with a PX330 plasmid, which may persist past the 1-cell stage and thus lead to low levels of mosaicism.      

Based on the imperfect controls I used, I would not take the TIDE quantitation of allele fractions literally.   However the qualitative results were pretty good and I wasn't able to find anything manually that TIDE didn't.  Also, this is a very fast analysis if you are performing sequencing on the PCRs anyway.   Moreover, it's easy to apply this analysis to PCRs on transfected pools of cells to measure CRISPR mutagenesis.  I'm looking forward to trying TIDE in this context as well.

Easy quantitative assessment of genome editing by sequence trace decomposition.  Eva K. Brinkman, Tao Chen, Mario Amendola and Bas van Steensel.    Nucleic Acids Research, 2014, Vol. 42, No. 22 e168

Watch Dr. Jennifer Doudna's Vanderbilt Flexner Discovery lecture on #CRISPR online: tinyurl.com/nwbeon7

Vanderbilt has set up a nice site for viewing of the Discovery lectures, so check out the link to hear and see Dr. Doudna's talk from last week.   The audio/video was pretty good - this is what we saw in the overflow room (which also overflowed!).  A great talk by Dr. Doudna.

http://mediasite.vanderbilt.edu/Mediasite/Play/b8701c579ec6438abcc0188275efa16a1d

Jennifer Doudna to give Vanderbilt Flexner Discovery Lecture Thurs. Jan. 8. #CRISPR http://tinyurl.com/mr927fk

Vanderbilt peeps, check out Vanderbilt's Flexner Discovery Lecture home page to view the announcement for Dr. Doudna's seminar this Thursday at 4 pm, 208 Light Hall.