#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.

Qin et al.  Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease.  Genetics, 2015 v.200 (2) pp. 423-430.

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.   

#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.

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.  

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.

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 18^th base on the protospacer strand and the 23^rd 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.

Link to a timeline of recent discourse on #CRISPR human germline gene editing.

I've started a separate blog about human germline gene editing.  Hope you like it.  My first post is a list of links to recent news items on the topic.

Webtool link for getting Xu et al #CRISPR target scores for your sequence of interest.

A followup to yesterday's post about the Xu et al paper,  Sequence determinants of improved CRISPR sgRNA design:   They have also kindly made a public webtool for generating CRISPR scores with their model.   It's a cut-and-paste that accepts up to 10000 bases.   Simple and quick.    

Of course, their source code is available too in their supplemental material and here.