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).
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.