CRISPR/Cas9: a potential cure for genetic diseases?

By Sabino Méndez Pastor

On October 7, 2020, the Royal Swedish Academy of Sciences awarded the Nobel Prize in Chemistry to Emmanuelle Charpentier and Jennifer A. Doudna “for the development of a method for genome editing”. This represents another example of how scientific research can take unpredictable turns that lead to surprising discoveries. When Dr. Charpentier started her own research group in 2002 to study how the genes of Streptococcus pyogenes bacteria are regulated, no one may have imagined that she would end up discovering the CRISPR/Cas9 genetic scissors: a powerful gene editor that is used to investigate the functions of different genes, to develop new cancer therapies and even to attempt to cure inherited diseases. Still, Dr. Charpentier always looked for the unexpected and this allowed her to develop with Dr. Doudna a technique that is revolutionising the life sciences. As she quotes Louis Pasteur “Chance favours the prepared mind” (Royal Sweedish Academy of Sciences, 2020).

But how does this promising genetic tool works? CRISPR/Cas9 is originally part of the Streptococcus’ immune system against viruses. When a virus infects a bacterium, it introduces its harmful DNA into it. If the bacterium survives the infection, it inserts a piece of the virus’ DNA in the CRISPR section of its own genome. CRISPR or clustered regularly interspaced short palindrome repeats are repetitive DNA sequences present in bacteria and archaea. Between these repetitions are found unique, non-repetitive sequences that match the genetic code of various viruses. These correspond to the pieces of viral DNA inserted into the bacterial genome as a memory of the infection. In addition, bacteria have CRISPR-associated (cas) restriction enzymes specialised in unwinding and cutting up DNA. In Streptococcus bacteria, CRISPR DNA is used to form CRIPSR/Cas9 complexes that include an RNA molecule complementary to the genetic sequence from a particular virus. If the bacterium is reinfected by that virus, the genetic scissors will immediately recognise it and disarm it by cleaving its DNA (Royal Swedish Academy of Sciences, 2020).

Thanks to the work of Charpentier and Doudna, researchers can now use this defence mechanism to edit DNA with more precision and greater ease than previous gene editing technologies. First, a guide RNA that matches the DNA sequence where the modification is to be made is artificially constructed and linked to a Cas9 protein. The guide RNA molecule directs Cas9 to the target DNA sequence where it produces a double-strand break. This is naturally followed by the recruitment of a DNA repair mechanism, either non-homologous end-joining (NHEJ) or homology directed repair (HDR). In the case of NHEJ, the ends at both sides of the cleavage are directly ligated back together. This process usually results in small insertions or deletions of DNA at the break that silence the gene. The HDR mechanism uses homologous sequences found in sister chromatids, homologous chromosomes, or extrachromosomal DNA as templates to repair the break more precisely. A DNA template can be artificially designed and introduced into cells as a template for the HDR, leading to defined genomic changes in the targeted cell (Royal Swedish Academy of Sciences, 2020). 

The precision of CRISPR/Cas9 genetic scissors makes them a good candidate to make a dream come true – curing inherited diseases. An excellent example of this are the numerous studies that are being conducted to identify possible genome-editing treatments for sickle cell disease (SCD). SCD is an inherited monogenic disorder that causes serious mortality and morbidity worldwide. It is caused by the substitution of a single nucleotide in the beta globin gene. This mutation leads to the abnormal aggregation of haemoglobin forming stiff rods within red blood cells that give them a sickle shape. Sickle cells often block small capillaries, slowing or even stopping the blood flow. Obstruction of vessels can result in extreme pain, stroke and spleen rupture which can cause internal bleeding and if untreated, death. 

At the moment there is no cure for SCD and only two medications have been approved by the U.S. Food and Drug Administration to lessen disease severity (Demirci et al., 2019). However, as SCD is caused by a localised mutation in a single gene, CRISPR/Cas9 has brought the possibility of using gene editing to cure the disease. Potential methods for gene therapy include modifying γ-globin regulatory elements to enhance foetal haemoglobin (HbF) production and direct gene correction of the SCD mutation. 

HbF is the predominant type of haemoglobin after the first trimester of gestation and is replaced by adult haemoglobin (HbA) around six months after birth. SCD symptoms decrease when HbF levels are high during adulthood, as seen in patients with hereditary persistence of foetal haemoglobin (HPFH). Gene edition of transcriptional regulators has been identified as a method to stimulate HbF production in SCD patients (Demirci et al., 2019). For instance, Weber et al. (2020) used CRISPR/Cas9 to generate insertions and deletions that disrupt the binding sites for the repressors BCL11A and LRF in the HBG promoters of the γ-globin gene of haematopoietic stem/progenitor cells (HSPCs). As γ-globin is part of HbF, the modifications made in either of the binding sites induced significant HbF production and corrected the sickling phenotype of red blood cells in vitro. Li et al. (2020) went further as they combined the disruption of the BCL11A binding site using CRISPR/Cas9 with the addition of γ-globin genes by SB100x. In order to avoid transplant-related toxicity, an HDAd5/35++ adenovirus vector was used to introduce these genetic modifications into HSPCs of sickle cell disease mouse models. The combination of the two genetic therapies resulted in a complete phenotypic correction of sickle cell disease. 

As the pathologic mutation for SCD is already clearly identified, another promising approach consists in directly correcting it using the CRISPR/Cas9 genetic scissors. This procedure was employed ex vivo by Dever et al. (2016) in patient derived haematopoietic stem cells (HSCs). Then, the modified HSCs were transplanted into immunodeficient mice. Corrected HSCs effectively differentiated into red blood cells that expressed adult β-globin mRNA, confirming intact transcriptional regulation of the edited gene.

In conclusion, CRISPR/Cas9 genetic scissors constitute a revolutionary tool that allows researchers to hope for a cure for genetic diseases. Many studies have shown that this technique can be used to correct the SCD mutation or its effects in ex vivo cell culture conditions and in mouse models. However, obstacles related with the efficiency and specificity of the editing, delivery of the modification, and immune responses against it, persist. Besides this, polygenic diseases caused by mutations on many genes represent another challenge for gene therapy due to their complexity. The cure for genetic diseases using gene therapy is moving closer to reality but questions remain to ensure it as a feasible, safe, and lifelong curative option. 


The Royal Swedish Academy of Sciences (2020) Genetic scissors: a tool for rewriting the code of life. Available from: [Accessed 23rd October 2020]

The Royal Swedish Academy of Sciences (2020) A tool for genome editing. Available from: [Accessed 23rd October 2020]

Demirci, S. et al. (2019) ‘CRISPR/Cas9 for Sickle Cell Disease: Applications, Future Possibilities, and Challenges’, in Advances in Experimental Medicine and Biology. Springer New York LLC, pp. 37–52. doi: 10.1007/5584_2018_331.

Dever, D. P. et al. (2016) ‘CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells’, Nature. Nature Publishing Group, 539(7629), pp. 384–389. doi: 10.1038/nature20134.

Li, C. et al. (2020) ‘In Vivo HSC Gene Therapy Using a Bi-modular HDAd5/35++ Vector Cures Sickle Cell Disease in a Mouse Model’, Molecular Therapy. Cell Press. doi: 10.1016/j.ymthe.2020.09.001.

Weber, L. et al. (2020) ‘Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype’, Science Advances. American Association for the Advancement of Science, 6(7), p. 9392. doi: 10.1126/sciadv.aay9392.

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