CRISPR Gene Editing in Mice and Rats

• Design and synthesis of CRISPR/Cas9 reagents
• Injection into embryos or cell transfer into blastocysts
• Reimplantation into foster female
• Husbandry and weaning
• Sample collection for genetic testing
• Full health report
• Delivery of transgenic mice

• Package I services
• Breeding to F1 generation
• Sample collection of up to 50 mice
• Delivery of heterozygous F1 mice

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Modifications in disease models that used to take months or years, can now be achieved in several weeks.

In most areas of biology, the more we learn, the more we realize how complicated the systems actually are. Modelling complex human diseases requires complex genetics: one might need to remove the normal mouse gene, then add in multiple human disease genes. As the field discovers more genes affecting outcome, the models necessarily get even more complex. In areas with diverse genetic modifications, like cancer, the extent of genetics required to properly unravel a problem can quickly become overwhelming. Here at Charles River, we are looking at several approaches to attain better research models, and that is where new genome engineering tools like CRISPR become so powerful. Check out this webinar to learn more.

There are two main ways to edit a genome. The classical approach used homologous recombination to trick a cell into adopting something close to normal sequence. To knock in a couple 100 bases with your mutation, you would need to include thousands of bases that exactly match normal sequence, on both sides. Even then the efficiency was quite low (there’s a reason we used 384 well plates)! Building those large targeting vectors, coupled with the low efficiency makes the process relatively slow.

The newer approach is to cause specific DNA damage (double strand breaks or DSB) and let the cell repair them naturally. In principle, anything that could cause a DSB would work. In practice, getting breaks exactly where you want them was very challenging, and time consuming. Before CRISPR, tools available were cumbersome, expensive or difficult to readjust. You might need to re-engineer most of the protein to target a new sequence. 

CRISPR allows modifications that previously took months or years to be achieved in weeks. The endonuclease (Cas9) is conserved and a short RNA guide can specify targeting. In the past 10 years, the RNAi (RNA interference) field has taught us a great deal about targeting sequences with small pieces of RNA, and much of that knowledge can be applied to CRISPR.

Utilizing CRISPR, transgenic mice can be generated faster than ever before and with great precision. Through directly injecting a one-cell embryo CRISPR can modify the entire animal while it is only a single cell. This virtually guarantees germline transmission (your modification passing on to the next generation), which is frustratingly left to probability when you use embryonic stem cells. Knocking out a single gene is straightforward, and while knock in and conditional mutations are more complex, a single microinjection session should generate the mice needed to found your new line.

I had the privilege of attending the Keystone Symposium on Precision Genome Engineering to present a poster and hear about the leading edge of technology in the field. CRISPR has accelerated the rate of progress in genome engineering to a truly dizzying pace.

Investigators are using CRISPR-based screens to look for targets driving disease, and in some cases even to look for mutants of a disease gene that lessen severity. Re-breaking a broken gene to fix it wasn’t the only outside-the-box idea that is now readily accepted. CRISPR is so good at targeting a specific sequence that some groups are using a ‘dead’ nuclease (one that can target but no longer cuts) tethered to other proteins that can turn genes on or off. This has been termed CRISPRa and CRISPRi (to activate or inhibit, respectively) and is being used to tune gene expression. Other reports were on ways to further increase CRISPR’s accuracy, allow broader range of targeting and even going back to microbial genomes to mine for the next endonuclease we can use as a tool.

Our poster, a collaboration between Charles River’s Leiden and Wilmington sites, laid the proof of concept groundwork for offering our new Model Creation service. We knockout lines using CRISPR through both indel formation and by knocking in a small stop cassette. In one experiment we show we can utilize both main repair pathways. We’ve also replicated the work through direct embryo injection and by modifying ES cells to cover all our bases (some constructs are complicated enough you would still want to start in ES cells). And we will continuously look for ways to improve our process.

One interesting challenge with CRISPR is the efficiency of editing. Usually when talking about efficiency, the problem is with it being too low. While that is the case with knock in constructs requiring homology directed repair (HDR)—there were plenty of talks about increasing HDR efficiency at Keystone—I’m talking about mosaicism.

It turns out that CRISPR often works so well that trying to modify a target gene will actually modify both copies! Every cell gets two copies of each gene (one from each parent), so the concern is that when you inject an embryo with CRISPR your target gene will be modified twice (once on each copy). This is called mosaicism and can be a little troublesome when one copy is the intended variant and the other is not, especially when those variants could vary by a single nucleotide. Carefully screening animals is critical, and thankfully our Genetic Testing Service has assays sensitive enough to find those subtle variations. Once you know which mice might be your founder, one more round of breeding can segregate those mosaic variants and you have your new line!

When I left academia years ago, it was to work with tool builders, optimize techniques and find better methods. I knew my personal impact on medicine would be greater if I helped drive the research of others. I am glad to be at Charles River, where we leverage powerful tools like CRISPR to accelerate research.

If you are interested in learning more about CRISPR, check out these recent articles in Eureka on humanized mice and the need to be prudent in using CRISPR.

Innovations in breeding and genotyping are helping refine and reduce the use of research animals.

Transgenic models are vital to research but generating them takes work—and a considerable number of animals. Moreover, when you breed a genetically modified animals it often results in ‘surplus’ animals.

However, scientists are devising ways to both refine and reduce the use of genetically-altered animals. Sara Wells, Director of the UK’s Mary Lyon Centre, which generates mice as models of human disease and is one of the hubs for the International Mouse Phenotyping Consortium, says a number of innovations have helped reduce genotyping and breeding of animals, such as strict colony management and different breeding schemes. At the same time, gene editing techniques like CRISPR/Cas9 is, ironically, both helping reduce the number of genetically altered animals in some situations, but increasing them in others. Nonetheless she sees a lot more attention being paid by researchers to the 3Rs than 25 years ago. “I see a real difference in new researchers, for whom the 3Rs is not an additional thought but really is really central to their studies,” she says.

How are innovations in breeding and genotyping helping you meet the 3Rs objectives? 

SW: There have been many technological advances in the last few years which have greatly helped us refine our breeding and genotyping. In terms of breeding, we use genetically controlled inbred lines (only bred for five generations prior to restocking to ensure that we are not susceptible to the effects of genetic drift.) We also use strict colony management regimes to reduce the number of static matings (where the male is left in so that females are kept breeding constantly) and cryopreservation to ensure that we don’t produce animals we don’t need.

On the genotyping front, the development of blastocyst and sperm genotyping has greatly reduced the number of embryo transfers we need to perform for quality assessment. At MRC Harwell we have been concentrating very hard on quality management; that is always using allelic-specific genotyping (not an assay for a generic sequence such as cre or neo).

As more research laboratories begin using CRISPPR/Cas9 to create their transgenic animals, do you think we’ll see a spike in animals being used in studies or a drop? 

SW: I think in individual studies there is a great opportunity for reduction. For some alleles (such as indels and point mutations), CRISPR/Cas9 is proving much more efficient in terms of generating mice carrying these modifications. This technology will allow us to develop more refined lines with genetic alterations which are more faithfully reflecting human diseases or much more specific models for discovery research (such as new recombinase lines or cell-specific reporters). Although there is likely to be a reduction in the generation aspect of these projects, the utility of the lines will increase. However, better preclinical models or more specific genetic models are likely to be more widely utilized and laboratories and may result in a concomitant increase in animal usage.

The major development which really will have an implication and could lead to an increase in GA animals are the large human genomic projects, sequencing hundreds and thousands of human genomes around the world and the ideas developing around precision or personalized medicine. It is inevitable that this will lead to an increase in demand for genetically altered animals (of many species) in an attempt to develop experimental models for investigating the mechanisms of many monogenic and polygenic genetic diseases.

What sorts of 3R innovations do you hope to see in the next 50 years? 

SW: I think there is a long way to go in the development of efficient genome editing technologies, both in terms of germ line changes and somatic changes. This has potential to impact on all 3Rs- refining existing models and inbred backgrounds, reducing the number used in generating new GA lines and better ways of making genetic changes in vitro could replace some of the in vivo work.

Along with an increase in sophistication in genome editing we also need to increase the sensitivity and reproducibility of in vivo phenotyping tests. The enormous progress in the electronics fields will allow more automated testing, in home cage environments and using telemetry; these really will increase the robustness and complexity of the data we are able to gather from GA lines.

Are the 3Rs still an important guideline for animal research today? Why? 

SW: Absolutely, they are the guiding moral principles. Just because we can, does not mean we should. Everyone has a moral obligation to seek alternatives for their in vivo experiments, use the least number of animals possible (although ensuring statistical robustness) and perform their experiments by methods which cause the least suffering. It is not a right to work with animals but a responsibility we all need to take very seriously.

How do you think the 3Rs has changed animal research in the last 50 years. 

SW: I can’t comment on 50 years (I am not that old!) but definitely over the last 25 years the 3Rs has become much more prominent in discussions around experimental design, scientific justifications and the development of refined methods. I still think that there is work to do in all establishments as we try to keep up to date and contribute to advances in the 3Rs.

I see a real difference in new researchers, for whom the 3Rs is not an additional thought but really is really central to their studies. This is of course aided and supported by organizations like the NC3Rs in the UK which provides training and meetings on 3Rs themes. Lastly, over the last five years a fourth R of reproducibility has been the topic of many debates and we see more efforts towards standardization, validation and robustness of testing which is very welcome.

Between 3Rs is a Q&A series created by the Charles River Laboratories’ Eureka blog and ALN Magazine to highlight the importance of the 3Rs—replacement, reduction, and refinement—as guidelines for ethical animal use in biomedical research. If you are interested in being a part of the series, contact [email protected] or [email protected].

With almost unlimited genome engineering opportunities, CRISPR holds great promise for transforming drug discovery and development.

The power of the CRISPR technology lies in its unprecedented ease and control when editing the genome. When you think of the drug development pipeline there are multiple steps that can benefit from this emerging technology.

In early discovery, CRISPR screens can reveal new drug targets and cell-based models can be engineered to mimic disease to increase translatability when searching for new compounds and going down the path of lead optimization and delivery of a candidate compound. But it does not stop here. Further preclinical development can be enhanced by CRISPR by applying the technology in in vivo target validation or generating new in vivo disease models with improved clinical relevance. Moreover, developments are ongoing at various CRISPR biotech companies to provide CRISPR-based gene therapy solutions for genetic diseases. The simplicity of the system, the accessibility to researchers, and the relatively quick way of genome engineering hold real promise for accelerating drug development and reducing attrition rates of compounds.

There is still a whole field out there to explore in using CRISPR screens for discovery of new disease targets. So far most of the publications are describing pooled screening in the oncology field. Pooled screening requires relatively clear-cut readouts, such as cell proliferation, cell death or sortable marker proteins. However, in other disease areas often more complex functional readouts are needed for a meaningful outcome of the screen. Arrayed screening allows for phenotypic readouts that are usually not amenable to a pooled approach, including high content analysis. Arrayed CRISPR libraries are now emerging with the guide RNA being either expressed from lentivirus or produced as a synthetic molecule. CRISPR faces the same challenges as RNA interference, where delivery is key to efficient screening. Using CRISPR screens in drug development is foreseen to grow now that CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens are coming into play, with the latter one giving the advantage of screening for gain-of-function.

In target validation the CRISPR technology has the advantage over RNAi to fully abolish gene expression as opposed to the transient and incomplete silencing of the target as achieved by siRNAs. This also has a profound impact on rescuing the phenotype to confirm target specificity by adding back methodology, which will give far better interpretable results in case of a complete knockout by CRISPR as opposed to an incomplete silencing by RNAi. Therefore, it is most likely that CRISPR-mediated knock-outs and knock-ins will be replacing the current target validation methods in the near future. Delivery systems such as viruses even allow for CRISPR-mediated target validation in a broad spectrum of primary cells that are usually more difficult to edit using conventional transfection systems.

The relative ease of engineering with CRISPR will certainly contribute to the development of more complex cellular assays with improved predictability for drug therapies. Also promising is the stem cell technology maturing in parallel with the CRISPR field. Genetic disorders can be mimicked by introducing genetic defects in stem cells, which can be subsequently differentiated into disease-relevant cell types.

Who would have thought 10 years ago that a revolutionizing technology such as CRISPR would re-shape the landscape of biomedical research? Yet this is the fortunate position we are currently in as researchers. It is an exciting time to be working in this rapidly expanding field and the future will tell us if the CRISPR promise in drug discovery can be fulfilled.