CrispR technology: Designer babies and Swine Donors

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The last decade has seen tremendous progress in ‘gene-editing’ techniques to make them more accurate, fast and replace the potential hit-and-miss methods of genetic engineering over the past few decades. The new technique called CrispR (pronounced Crisper!!) that emerged from pioneering work in bacterial cells in the early 2000s has evolved over the years to find applications in biotechnology, fundamental research and medical science.

The preliminary CrispR toolkit involved a RNA molecule called gRNA (guide-RNA) that is homologous to the area of DNA you wish to modify and a nuclease Cas9. The gRNA, as the name suggests, guides Cas9 to the region of interest to create a double strand break (DSB). The native cellular repair machinery: NHEJ (Non-homologous end joining) or HR (Homologous recombination), then repairs and introduces the necessary changes using a template DNA. Once the basic methodology of this technique was established, CrispR was used widely for gene-editing in almost every living organism from cells to bacteria, insects to plants and animals to now human embryos Doudna & Charpentier 2014; Sander & Joung 2014).

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Two studies published in the recent months show promising results with CrispR-mediated gene editing in embryos. The first study at Oregon Health & Science University (OHSU) and Salk Institute in the US, focuses on using CrispR for genome-correction to avoid transmitting genetic disorders in human embryos (Ma et al. 2017). The authors were keen on targeting genetic disorders that manifest in late-adulthood such as MYCBP3 gene mutation, which leads to hypertrophic cardiomyopathy (HCM). This genetic disease occurs in one in 500 and is a common cause of death in young athletes. The scientists CrispR-ed the mutated allele of MYCBP3 in embryos from IVF donors and replaced it with the wild-type maternal allele instead of a synthetic DNA template. By injecting the Crisp-R-Cas-9 cocktail along with the sperm (from a HCM male patient) into a wild-type oocyte (source of maternal DNA), they achieved increased correction rates and avoided mixed populations in the resulting embryos. Additionally, whole genome sequencing identified very few off-target effects of the gene editing, promising a potential break-through in germ line gene therapy. For ethical reasons these embryos could not be implanted into a uterus to develop a baby.

Yet another promising venture for CrispR was in the organ-transplant arena, where scientists in China have successfully CrispR-ed the retroviral gene in pigs (Niu et al. 2017). The study has attracted huge media attention as it promises xeno-transplants from pigs to humans without the fear of an immune attack. Traditionally such transplants are rejected in humans, as the PERV gene from pigs leads to retroviral infection in humans. The scientists attempted to target 25 copies of the PERV-gene (Porcine Endogenous Retrovirus) all at once using CrispR in adult cells, that would be later fused with ova (female reproductive cell) to grow into embryos and implanted in sows. Although, they met with disappointment in their initial trails due to ‘shredded-genomes’ in the targeted cells, Laika – the PERV inactivated piglet, was born after using a specific cocktail that kept the Crisp-R cells alive despite the aggressive gene-editing. Many other similar studies are underway exploring organ transplants from swine donors, by modifying their genome to ‘humanise’ them for successful transplants (Petersen et al. 2016; Martens et al. 2017) . While controversies continue in the field, if gene editing is required to replace anti-viral drugs in such a transplant, CrispR surely shows immense potential in the clinic.

Owing to the recent discoveries this gene-editing tool is now under the scrutiny of ethical committees and policy makers, as they fear a domino effect and the advent of ‘designer babies’. Nature, earlier this month published a report that marked down this technology due to the unintended mutations it caused in-vivo in a mice experiment (Schaefer et al. 2017). The study asserts the importance whole genome sequencing if these tools were to be applied in ‘real-people’. Although geneticists are not unaware of the potential pitfalls in genome editing, if Crisp-R will survive these hurdles only time will tell.

Blog written by Nisha Peter


Doudna, J.A. & Charpentier, E., 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science (New York, NY), 346(6213), pp.1258096–1258096.

Ma, H. et al., 2017. Correction of a pathogenic gene mutation in human embryos. Nature, 72, p.1117.

Martens, G.R. et al., 2017. Humoral Reactivity of Renal Transplant-Waitlisted Patients to Cells From GGTA1/CMAH/B4GalNT2, and SLA Class I Knockout Pigs. Transplantation, 101(4), pp.e86–e92.

Niu, D. et al., 2017. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science (New York, NY), p.eaan4187.

Petersen, B. et al., 2016. Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes. Xenotransplantation, 23(5), pp.338–346.

Sander, J.D. & Joung, J.K., 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology, 32(4), pp.347–355.

Schaefer, K.A. et al., 2017. Unexpected mutations after CRISPR-Cas9 editing in vivo. Nature Methods, 14(6), pp.547–548.

Fabulous Fluorine in Medicinal Chemistry

Since the FDA approved the steroid fludrocortisone, the first fluorine-containing drug, in 1955, the number of fluorine-containing drugs appearing on the market has rapidly risen to approximately 25% of drugs.1 These include the blockbuster drugs Prozac, Lipitor and Prevacid, Fig. 1. This may seem like an unusual trend considering fluorine-containing natural products are quite rare, so why is it that fluorine is so abundant in drugs? When you consider the unique chemical properties that fluorine has, the reason for a drug designer’s love of fluorine becomes clearer.

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Fig. 1: Examples of fluorine-containing drugs

Fluorine is a small, highly electronegative and lipophilic atom. The incorporation of fluorine into a molecule is an increasingly popular strategy to improve drug potency. Fluorine substitution can enhance potency and impact target selectivity by affecting pKa, modulating conformation, hydrophobic interactions and lipophilicity. Fluorine is also frequently used to improve drug metabolism. More recently, radiolabelled fluorine drugs have been used in positron emission tomography (PET) for imaging and diagnosis purposes in medicine.2

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Fig. 2: Effects of fluorine in medicinal chemistry3

Fluorine is much more lipophilic than hydrogen, so incorporating fluorine atoms in a molecule will often make it more fat soluble. This means permeability across membranes is increased resulting in a higher bioavailability. Because of the strong electron withdrawing ability of fluorine, it’s inclusion in a molecule has a very strong effect on the acidity or basicity of proximal functional groups. Altering the pKa can strongly modify the binding affinity and the pharmacokinetic properties of a pharmaceutical agent. Often, fluorine is introduced to lower the basicity of a compound which aids in permeability.4

Fluorine can play an important and unique role in influencing molecular conformation. Sterically, fluorine is similar in size to a hydrogen atom, but the high electronegativity of fluorine results in a highly polarised C-F bond with a strong dipole moment and a low-lying C-F anti-bonding orbital available for hyperconjugative donation.2 This can cause fluorine-containing molecules to adopt a different preferential conformation compared to the non-fluorinated molecule which may result in increased binding affinity to a receptor.

One of the major challenges in drug discovery is that of low metabolic stability of compounds. Lipophilic compounds are susceptible to oxidation by liver enzymes, in particular cytochrome P450. Fluorine substitution at the metabolically labile site or at adjacent sites to the site of metabolic attack is a common strategy to improve metabolic stability. The inductive effect of fluorine should result in decreased susceptibility of adjacent groups to metabolic attack by cytochrome P450. Additionally, fluorine can modulate lipophilicity and restrict conformation, which may afford improved metabolic stability.3

PET imaging using 18F tracers is a rapidly developing area in medicinal chemistry. PET scans show biological processes which can give invaluable metabolic information. PET is used as a diagnostic tool, particularly in oncology, but also as an in vivo pharmacological imaging tool in drug development, especially in the areas of biodistribution and drug occupancy studies.4

One drawback of introducing fluorine substituents into drugs is that fluorination can be rather difficult and many processes create challenges in the manufacturing process. However, numerous new, safe and mild fluorinating reagents have been invented in recent years, many of which are commercially available, making the process much simpler.5,6

Blog written by Catherine Tighe


  1. Wang, J.; Sánchez-Roselló, M.; Aceña, J. L.; del Pozo, C.; Sorochinsky, A. E.; Fustero, S.; Soloshonok, V. A.; Liu, H. Chemical Reviews 2014, 114, 2432.
  2. Zhou, Y.; Wang, J.; Gu, Z.; Wang, S.; Zhu, W.; Aceña, J. L.; Soloshonok, V. A.; Izawa, K.; Liu, H. Chemical Reviews 2016, 116, 422.
  3. Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Journal of Medicinal Chemistry 2015, 58, 8315.
  4. Purser, S.; Moore, P. R.; Swallow, S.; Gouverneur, V. Chemical Society Reviews 2008, 37, 320.
  5. Yerien, D. E.; Bonesi, S.; Postigo, A. Organic & Biomolecular Chemistry 2016, 14, 8398.
  6. Campbell, M. G.; Ritter, T. Organic Process Research & Development 2014, 18, 474.



Letting the public loose in our labs

On Sunday 25th June 2017, University of Sussex hosted its first Community Festival. The local public were invited to explore the Falmer campus and get involved with a number of hands-on activities across the various schools and departments. There were taster activity sessions at the Sussex Sport facilities, nature walks around the campus, live jazz, talks and more. Thousands of people turned up for the day (1).


Sussex Drug Discovery Centre was represented on the programme by a delegation of our group – Trudy Myers (SDDC Co-ordinator), Jess Booth (Assay Development and Screening Biologist), Kay Osborn (Biology Technician), Lucas Kraft (PhD Student) and myself. Those who signed up to our activity were taken on a whistle-stop tour through the drug discovery process.

First of all, Jess gave an overview of what the drug discovery process entails (see Figure 1) and where the SDDC fits into that process.

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Figure 1 : the drug discovery process (2)

After this introduction, our guests donned lab coats and safety glasses and were given a tour of the group’s protein purification suite. Kay explained how the term “protein” is used in the context of drug discovery to describe tiny molecular machines in our bodies that carry out a variety of functions, and in some cases, cause disease symptoms. She showed the group some incubating flasks containing E. coli which is often used to produce a large quantity of these disease-causing protein. Then she explained how that protein can be later isolated from the bacteria cells via a technique called FPLC (fast protein liquid chromatography) (3) which separates proteins out by their different sizes.

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Next, the newly-briefed scientists were taken into the main biology lab where they were able to run their own biochemical assay. While some took to the pipettes quicker than others, all had fun pipetting reagents onto plates that had lots of different drug compounds in them to see if they got any hits – wells that turned pink indicated a successful compound that had bound to the protein and inhibiting its function while blue ones were unsuccessful ones.

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During the last part of our activity I showed our participants around the oncology chemistry lab. I pointed out many of the similar pieces of kit that they would find in their kitchen. We discussed how the results from the assay they had just run in the biology lab informs the work that is carried out by chemists and how we vary the procedures (or “recipes”) we use to make new drug compounds.

The feedback for the activity was overall very positive and our enthused guests went away knowing a lot more about the drug discovery process than when they arrived, which was our aim.

Fiona Scott is a first year PhD student at the SDDC.


  1. University welcomes local residents for fun-filled day of discovery. University of Sussex News. [Online] [Cited: 1st August 2017.]
  2. NMT Pharmaceuticals. [Online] [Cited: 1st August 2017.]
  3. Dermot Walls, Sinéad T. Loughran. Protein Chromatography Methods and Protocols. New York : Humana press, 2011. pp. 439-447.