Wednesday, February 24, 2016

My recommendations for mixing and diluting #CRISPR gRNAs, mRNAs, and HDR oligo DNAs for mouse zygote injections.

It's super convenient to outsource CRISPR reagent construction to vendors (shout out to my friends at Sigma-Aldrich) and also to outsource generation of CRISPR mice to your friendly neighborhood transgenic core (shout out to the Vanderbilt TMESCSR).   However there still is that step where you may have to actually handle the RNAs/DNAs and mix them together, before handing them off to your friendly transgenic core staff.  I often get asked how I do this.  It's nothing tricky but it does involve RNA, so of course, be clean and careful.  Here are my current recommendations based on receiving RNAs shipped on dry ice from Sigma-Aldrich.

Recommendations for mixing and diluting CRISPR gRNAs , mRNAs, and HDR oligo DNAs for mouse zygote injections.  

Doug Mortlock Feb 2016

•A chart with the recommended final concentrations is found on the TMESCSR website ( https://labnodes.vanderbilt.edu/resource/view/id/11363 ) and is reproduced here.

•Sigma-Aldrich ships the RNAs at slightly higher concentrations than shown below.  Cas9 mRNA is provided at 500 ng/ul and gRNAs at 200 ng/ul.   

•I do not further clean up or process the RNAs in any way prior to dilution.   However, I do carefully re-precipitate the HDR oligos and resuspend them in sterile RNAse-free water.    This can remove some contaminating non-DNA material that is sometimes present in lyophilized oligos as shipped from the vendor.

1.     To prepare “N” injection days worth of injection mixture aliquotes, prepare N+1 tubes as follows.  Use clean, RNAse-free 1.5 ml microfuge tubes, sterile RNAse-free water, and RNAse-free arosol barrier tips.   Pre-rinse each tube by adding 1 ml of the water, vortex, and dump out all the water. Spin down the tubes briefly and remove any lingering dregs of water with a pipette tip. Place all the tubes on ice.

2.     Mix the RNAs, water (and DNA oligo as needed) to create a mix with the correct final concentrations of all reagents and volume that is equal to at least (N x 25) + 5 µl.  Mix briefly by flicking the tube gently (do NOT vortex). 

3.     Spin the mixture at full speed in microfuge for 1 minute. This is to pellet any small particulates – even if none are visible to the eye, before or after this step!

4.     Drawing from the top of the mixture volume, remove 25 µl and aliquot to one of the tubes.  Repeat for each aliquot.  There will be ~5 µl left over.  This hopefully has concentrated any particulate material (that might clog injection needles) and is discarded as a sacrifice to the CRISPR gods.   Although it is NOT usually visibly apparent that there are ANY particulates present, this step is added because it is TYPICAL for the injection technicians to have needle-clogging problems with CRISPR injections.  This reduces embryo survival.   While this pre-spin step may not always solve the problem it may help and is easy to do.

5.     Clearly label the aliquot tubes and bring them on ice to the transgenic core. They should be stored at -20˚ until injection date.   


The chart below was culled from our TMESCSR recommendations as of Feb 2016.  I know 'cause I wrote 'em.

Create the required mixture of components so that it has the final concentrations shown below.
The "min volume to bring to Core" is specific to the Vanderbilt's core's preferences.  So don't use this to argue with your core's staff about what is necessary.  They get to decide that for themselves!



Experiment type
Final conc. Cas9 mRNA 
Final conc. all gRNAs
Final conc. all ssDNA donor oligos

Buffer

Min. volume to bring to Core
Knockout experiment, RNA reagents only

100 ng/µl
50 ng/µl
-
Sterile, RNAse-free water
25 µl
PER INJECTION DAY
Knock-in experiment, RNA + ssDNA oligo(s)

100 ng/µl
50 ng/µl
200 ng/µl
Sterile, RNAse-free water
25 µl
PER INJECTION DAY
Requested deviations from these concentrations will require consultation and approval from the core manager.   The core staff may need to dilute the reagents more if injection problems arise (e.g. clogging).

REQUIREMENTS FOR PURIFICATION:
• We strongly suggest ssDNA oligos be re-precipitated before use to remove potential contaminants from the vendor.    See TMESCSR website for this protocol.

Thursday, January 28, 2016

For #CRISPR HDR, use donor oligos that are complementary to the "gRNA strand". A new paper shows why; see my blog post.

(ERRATUM:  I made on correction to this post on March 11 2016.  In the original version of the post I stated that the paper implied that the donor oligo should have "additional length of homology on the PAM-distal side as compared to the PAM-proximal side".  That was a mistake - it turns out the opposite was true.   The authors found that additional length of homology on the PAM-proximal side was favorable.  I got confused because the PAM in figure 3 is on the "bottom" strand, not the top, so the PAM-proximal side is to the left of the cut site in their oligo schematics.  Figure 3 has an "upside down" Cas9 icon in keeping with this fact.  Thank you Scot for letting me know about the error!).


After a long break from blogging... here's a nice nugget of insight about CRISPR-mediated homologous recombination.    Back in late 2014 I blogged about 
Optimal design of ssODNs (donor oligos) for #CRISPR - length and strandedness data? . In that post I pointed out a curious observation that HDR oligos work "better" when they are designed from the strand that is complementary to the protospacer/gRNA sequence.   This was somewhat counterintuitive to me, as one might think that in a complementary HDR oligo would tend to anneal to the gRNA, reducing its availability or kinetics somewhat and generally interfering with Cas9's job.   But empirically, this was not the case; complementary oligos work better.

Now, Richardson et al seem to have found an explanation. (Richardson et al, Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nature Biotechnology (2016, Published online 20 January 2016)). Turns out that following double-stranded DNA cleavage by Cas9, the first component of the DNA molecule that it releases is the 3' end of the DNA strand that corresponds to the protospacer/gRNA.  Quote: Hence, although Cas9 globally dissociates from duplex DNA in a symmetric fashion (Fig. 1c), it appears that the enzyme locally releases the PAM-distal nontarget strand after cleavage but before dissociation."  And here is a nice diagram from the supplemental material of the paper that shows how this strand "breathes" after cleavage.  I thank the senior author, Jacob Corn, for graciously allowing me to reproduce the image here:




(OK - before moving forward, let's get clarity on the terms here; when we're comparing the two strands of the DNA containing the CRISPR target, the "target" strand is the DNA strand that directly will anneal to the gRNA.  Thus, the "target strand" is complementary to the gRNA.  The non-target DNA strand encodes the protospacer and the "NGG" PAM sequence.   Got it? )   

Read the above quote again.  The first bit of DNA that is released by Cas9 is the single-stranded 3' end of the non-target strand.  This immediately suggests 2 things:

1.  Cas9 releases the non-target strand before the target strand.  Thus the non-target strand is available sooner than the target strand to potentially engage with a complementary donor molecule and jump-start homologous recombination.      

2.  The 3' end of the non-target strand, which is "PAM-distal", is released first.  This also suggests that the design considerations for homology might be different for the PAM-distal and PAM-proximal sides of the cleavage location.

For this second point, the practical consideration is that commercially available ssDNA oligos are usually limited to 200 bases or less (depending on the vendor's capabilities; 120 base oligos can work well too).  So, we are limited in the length of homology we can actually apply to each side of the cleavage site.   Instead of centering the oligo (e.g. for a 120 base HDR oligo = ~60 bases of homology on each side) it may be better to skew the oligo design to have more homology on one side of the cut site versus the other.  That is what Richardson et al found - at least for one target they investigated in detail  (See Fig. 3c, d, e.)

Here's another thought. The authors note that Cas9 actually stays on the DNA for quite a while after it cleaves both strands - about 5 hours.    This might have something to do with why DNA repair takes significantly longer on Cas9-cleaved breaks than on breaks induced with radiation - Cas9 may just sitting there, sterically hindering the DNA repair proteins from accessing the free DNA ends at the break.  Perhaps, CRISPR mutagenesis efficiencies could be further enhanced by increasing Cas9's intrinsic off-rate?  On this note, another recent paper by Kleinstiver et al (Keith Joung lab) suggests that directed mutations can destabilize Cas9's non-specific interaction with DNA.   The authors note that this reduces off-target cleavage significantly while preserving on-target cleavage .   While they did not see dramatic increases in on-target efficiency, perhaps in some experimental contexts there might be?    Hmm.    

Friday, November 13, 2015

#CRISPR editing mouse embryos by direct zygote electroporation - no microinjection needed.

I’ve been intending to blog on this for a while now and finally got around to it.   This paper comes from a group at the Jackson labs; the first author is Wenning Qin, and the senior author is Haoyi Yang who also holds a primary appointment at an institute in Beijing.


In this paper they show that CRISPR-Cas9 reagents can be electroporated directly into mouse zygotes to generate gene-edited animals.  It’s not quite as efficient as direct injection into zygotes – but it’s not too shabby, considering the ease of the electroporation step.

To remind those who are unfamiliar, the main method to deliver DNAs or RNAs into mouse zygotes is through direct pronuclear or cytoplasmic injection, using ultrafine capillary needles.  It requires micromanipulators for the needles, carefully controlled pressure to deliver the injected material, and a quality inverted microscope with high-contrast optics.  Plus a steady hand and skill at injections.  In mammals, zygotic DNA transgenesis has generally required direct injection of DNA into the pronuclei (unless a retrovirus is used, which has its own drawbacks).  This was shown in papers from the early days of transgenic mouse research.  

Historically, electroporation has not been used for engineering mouse zygotes.   There are some good reasons for this.  First, zygotes have a zona pellucida surrounding the zygote itself.  This can be dissolved away rather easily with brief acid treatment – but without the zona, the embryos are sticky and much more difficult to handle.  Second, electroporation doesn't immediately transfer material into the zygote nucleus, and the chance of DNA integrating into the genome is very low.  In fact, even direct injection of DNA into the zygote cytoplasm does not yield transgenic mice efficiently – you’ve got to inject it into the pronuclei.    

Now, there are research applications for zygote injection apart than transgenesis.  You might just want to transiently express an mRNA in a zygote, for example.  Embryologists who work with zebrafish and xenopus will be totally familiar with this idea.  It’s not done frequently in mice but can be done.  I think the labs that ever do this with mouse embryos are hardcore enough they have access to microinjection equipment, and presumably haven’t bothered to try electroporation much - why would you, if you are all set up to perform the established method.

However…what if you either (1) want to try transgenic manipulation, but don’t have access to a microinjection apparatus, or (2) you just want to really streamline the labor involved?  Then electroporation might be useful…  Enter CRISPR, in which we actually do want to transiently express the reagents in zygotes.  At Jax they fall into the (2) category.

To develop this method, Qin et al. first confirmed previous reports that brief incubation in acid can be carried out to weaken the zona pellucida without completely dissolving it, while not affecting embryo viability.  Next, they tested electroporation parameters to optimize both the media/TE mixtures compatible with embryo survival and the maximum voltages the embryos could tolerate and still live.   Finally, they mixed acid-treated embryos with Cas9 mRNA plus guide RNAs for known pre-validated targets in the Tet1 or Tet2 genes and did electroporations.  Surviving embryos were either genotyped after in vitro culture, or transferred into recipient females and analyzed after birth.  

Bottom line: they could generate mutant animals at double digit percentages.   Not surprisingly, efficiency increased with higher RNA concentrations.  The final standard conditions involve at least 30 to 50 embryos per electroporation, in a total volume of 20 µl buffer/media with final concentrations of 600 ng/µl Cas9 mRNA and 300 ng/µl guide RNA.  Note that this requires a total of 12 µg Cas9 mRNA and 6 µg gRNA per batch of 50 embryos.   They also showed HDR is possible by co-electroporation with donor oligo DNAs.

So what is the efficiency?  Table 2 is a very nice comparison of microinjection vs. electroporation across ten genes.  Kudos to them for a nice big data set!  Good news: electroporation generated mutants for 5/10 genes tested.  However, 8/10 microinjections were successful for the same genes/reagents.   The overall rate of mutants was lower in the electroporation set as well; the average efficiency in the 5 successful electroporations was 20%, while with microinjections it was 42%.   Interestingly, the overall rate of embryo survival to birth was higher for electroporations – about double that of microinjections.   So in the end, the lower efficiency of mutant generation via electroporation might be pretty well balanced by the higher birth rate per embryo.

I’ll still note that zygote manipulation may not be for the faint of heart even if you don’t have a microscope/microinjection setup handy.   Remember that to make engineered live mice you have to transfer the manipulated zygotes back into a pseudopregnant recipient female mouse.  This requires microsurgery, people.     

However – maybe even this last step can be improved upon.   Takahashi et al have now reported that embryos can be electroporated – and CRISPR-mutagenized – without taking them out of the oviduct.    (Takahashi et al, Scientific Reports, June 22 2015 (5:11406).)  This still requires injection of the CRISPR RNA solution into the oviduct of a live mouse, but it’s probably easier than transferring embryos back in to the oviduct.   Hmm… maybe one doesn't need to pre-weaken the zone pellucida?  Their method requires rather high RNA concentrations, but bears promise.   


Wednesday, October 28, 2015

#CRISPR / gene editing featured at Festival of Genomics Nov. 3-5 in San Mateo. #genomicsfest

I will be participating in a CRISPR mouse editing workshop next week (Nov. 3) at The Festival of Genomics, in San Mateo, CA.  The full workshop title is "CRISPR/Cas9 Genome Editing Pipelines for Mice and Rats" and I'll be joined by Thom Saunders (University of Michigan) and Kevin Peterson (The Jackson Laboratory).  

The Festival of Genomics is a fairly new, recurring conference that has a lower registration fee than most traditional scientific-society meetings.  The speaker lineup for the meeting next week is pretty strong, with a strong biotech presence not surprising given the locale, but also strong plenaries from academics as well (Jennifer Doudna, of course; Carlos Bustamante, Manolis Kellis etc).  

Looks like Tues. Nov. 3 is mostly workshops, and Weds & Thurs Nov. 4-5 will feature plenary talks in the morning and then four concurrent tracks of sessions: Genome Editing, Genome Analysis, Genomic Medicine, and Data Analysis.    If you're in the SF bay area, check it out.

Friday, October 23, 2015

Updated guidelines for mouse #CRISPR injections in the Vanderbilttransgenic core.

I have revised the CRISPR guidelines on our Vanderbilt transgenic core's website.

 https://labnodes.vanderbilt.edu/resource/view/id/5265/collection_id/14/community_id/8

Basically it describes the basic information about what to do, and more importantly what to know, to get a CRISPR mouse project started through our core.   This may be most useful for my Vanderbilt peeps but others may find it interesting, as it gives some insight into how our core facility is communicating these sorts of guidelines to our users.  

These guidelines are not heavy on the up-front, nitty-gritty CRISPR design aspects as there are other places to find that stuff - for example, in the archives of this blog, as well as through the links I provide on the right side of this blog page to some helpful tools.  

Thursday, October 1, 2015

UK researchers ask you to submit your opinions about gene editing - link to web survey. #CRISPR

Dr. Lara Marks and Dr. Silvia Camporesi would like you to tell them your opinions about gene editing technology via their online survey.   What do you think? Let them know.  

Dr. Marks edits the hosting website, What is Biotechnology.   Dr. Camporesi is a bioethicist at Kings' College London.

Monday, September 28, 2015

Move over, Cas9: Cpf1 may be your new #CRISPR competition.

This past weekend at the Pilgrimage music festival in Franklin, TN I had the pleasure of seeing Weezer rock out, then I walked a few hundred yards to another stage to watch Wilco do the same.  These bands each had their own stage, but in the CRISPR world, Cas9 is a superstar that now has to share the stage with a newcomer:  Cpf1.  (Yeah, I know that's a goofy setup but it really was a good festival and it was on my mind.  Now on to the science.)

Last Friday Feng Zhang’s group published a paper in Cell that immediately grabbed a lot of attention, and rightly so.  They reveal that the Cpf1 class of CRISPR effector proteins may be an attractive alternative to Cas9.   Although Cpf1 has many similarities to Cas9, it has some significant differences that are very interesting – and could lead to improved efficiencies for some types of gene editing.  

Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System.  Zetsche et al., 2015, Cell 163, 1–13October 22, 2015  (Avail. online Sept. 25 2015).

Here is my summary…First, they reviewed some background on CRISPR systems in bacteria to cover the basis of the study.   There are two major classes of CRISPR systems based mainly on the proteins involved; the cleavage effectors of class 1 are complexes of multiple proteins, while the class 2 effectors are single proteins like Cas9.  Within class 2 there are two subtypes of systems: those with Cas9, and another that has Cpf1.   Cpf1 means “CRISPR from Prevotella and Francisella 1”.  Some bacterial species carry both Cas9-CRISPR and Cpf1-CRISPR loci in their genomes.   Like Cas9, Cpf1 has RuvC-like DNA cleavage domains but it lacks some of the other domain and neighbor-gene features of Cas9, so it’s clearly distinct in its evolution.    It’s a largish protein of ~1300 amino acids, similar in size to Cas9.

They picked the Cpf1-CRISPR gene system of Francisella novicida strain U112 to study first since there were clear homologies of Cpf1-CRISPR spacer sequences to various prophage in this species – further suggesting that Cpf1 is important in bacterial immunity and so it’s well adapted to slice and dice target DNAs.   (Sidebar: what’s F. novicida? A pretty rare human pathogen, originally isolated from the Great Salt Lake in Utah.  It’s related to the better known bug F. tularensis which is one of the most infectious pathogens known.)     

By transferring the F. novicida Cpf1-CRISPR gene locus into E. coli they quickly established that it prefers a  “TTN” PAM motif that is located 5’ to its protospacer target – not 3’, as per Cas9.  So right away it’s distinct in having a PAM that isn’t G-rich and is on the opposite side of the protospacer. 

Like Cas9, Cpf1 binds a crRNA that carries the protospacer sequence for base-pairing the target.  But for me the biggest surprise in the paper is that unlike Cas9, Cpf1 does not require a separate tracrRNA – in fact, there’s no sign of a tracrRNA gene at the Cpf1-CRISPR locus.   Thus, Cpf1 merely needs a cRNA that is about 43 bases long –of which 24 nt is protospacer and 19 nt is the constitutive direct repeat sequence.   This is very different than Cas9 – even by fusing the crRNA and tracrRNA, the single RNA that Cas9 needs is still ~100 nt long.

Furthermore, the Cpf1 crRNA does not have the long stemloop structure that is typical of RNAseIII-mediated processing to cut it out of its primary transcript.   It has a much shorter stemloop that is required for Cpf1 activity, however.   But surprisingly, Cpf1 itself is apparently directly responsible for cleaving the 43-base cRNAs apart from the primary transcript in the first place! This isn’t conclusively proven yet, but is pretty likely based on their experiments.   

Next, two more surprises comes from the cleavage sites on the target DNA.  The cut sites are staggered by about 5 bases.  This should create “sticky overhangs” that might be exploitable to enable gene editing via NHEJ-mediated-ligation of DNA fragments with matching ends.   And, the cut sites are in the 3’ end of the protospacer, distal to the 5’ end where the PAM is.    The cut positions usually follow the 18th base on the protospacer strand and the 23rd base on the complementary strand (the one that pairs to the crRNA).

They tested if they could inactivate the DNA cleavage domains via homologous mutations in codons known to do this in Cas9.  However, the resulting Cpf1 mutants don’t have “nickase” activity – they can’t cut either strand.   So it’s not clear that Cpf1 nickases can be made and in fact the authors suggest that the cleavage might require some sort of dimerization.  I can’t visualize how that would work yet but I’m sure it will be figured out in the near future…

Base substitution experiments then showed that, as per Cas9 CRISPRs, there is a “seed” region close to the PAM in which single base substitutions completely prevent cleavage activity.   Therefore, unlike the Cas9 CRISPR target the cleavage sites and the seed region do not overlap.  This immediately suggests a potential improvement over Cas9 in mammalian HDR-mediated repair efficiency.    This is because any initial cleavage events that might lead to “simple” NHEJ indels might still be substrates for cleavage  - and thus allowing additional chances for HDR-mediated edting to occur.  With Cas9, an indel mutation will almost always disrupt the target seeds and then it’s game over for HDR.     

Finally, they did the important work to screen various Cpf1 proteins from different bacterial species to see if any would actually work in mammalian cells.  This is because that despite codon optimization and attachment of nuclear localization signals, most of these bacterial proteins just don’t work right when you put them inside human or mouse cells.  Therefore they tested 16 different Cpf1 proteins.  Of these, for seven proteins they could identify PAM signatures using their E. coli assay.  They all had similar T-rich PAMs. 

Of these seven proteins, only two worked well in human HEK293 cells – AbCpf1, from an Acidaminococcus, and LbCpf1, which is from a Lachnospiraceae; interestingly these are apparently both anaerobic bacteria sometimes found in mammalian intestines.  Anyway these can both generate indels at specific targets in human cells at a rate similar to Cas9 – typically, 10-20% Surveyor assay numbers were observed, when they tested HEK cells following simple transfections.

Bottom line: Cpf1 may be the real deal as a serious competitor for Cas9.  Is Cas9 suddenly obsolete?  Hardly.  First, we don't yet know if Cpf1 is as specific as Cas9 – though there is every reason to think it may be.   So the off-target effects need to be carefully measured. Second, we don’t know how widespread the targeting efficiencies will be across sites (although the initial tests seem very promising).  Third, although Cpf1 may be better than Cas9 for mediating insertions of DNA, it’s not yet been shown if that is true.   However it may have some nice advantages over Cas9, not the least of which is that its guide RNA is only 43 bases long.  It will thus be feasible to purchase directly synthesized guide RNAs for Cpf1, perhaps with chemical modifications to enhance stability.

Probably, Cpf1 and Cas9 will both be in the spotlight for a long while to come.  Look for more Cpf1 papers to start coming out very soon and for Cpf1 plasmids to appear in Addgene.  Happy CRISPRing, everyone.