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European Biopharmaceutical Review

Science Fact

Last year’s discovery of a revolutionary gene editing tool created huge excitement in the biomedical industry. The CRISPR/Cas9 system – which enables the targeted modification of DNA in living organisms – offers greater efficiency, precision and flexibility than previous gene editing technologies – and with it, the potential to eradicate numerous diseases

There was once a time where gene editing – or genome engineering – was considered to be science fiction, due to its long and random process: it started with the deletion or insertion of a DNA sequence at a specific location, and increased its accuracy through a technique called homologous recombination. Because the desired recombination events occur very infrequently (1 in 106-109 cells), using this method presented enormous challenges to the large-scale application of gene-targeting experiments and, as a result, its application has been limited in most organisms (1,2).

DNA-Binding Proteins

Today, genome editing is based on the use of engineered or programmable nucleases composed of sequence-specific DNA-binding domains bound to a non-specific DNA cleavage module (3). These nucleases are guided to a specific sequence within the genome to induce a double-strand DNA break (DSB). When a DSB is generated, the cell’s intrinsic DNA repair system is activated and the genome is modified during the repair of the DSB. DSBs are typically restored by either non-homologous end joining or homology-directed repair (2). There are three main DNA-binding proteins that have been engineered thus far; however, each of these carry challenges with either specificity, flexibility or adaptability of gene editing (1):

● Meganucleases derived from microbial mobile genetic elements
● Zinc-finger nucleases based on eukaryotic transcription factors
● Transcription activator-like effector nucleases from Xanthomonas bacteria

Following the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) endonucleases-RNA-guided DNA endonuclease (Cas9) from the type 2 bacterial adaptive immune system, everything has changed and gene editing has become a lot easier.

System of Choice

In the CRISPR/Cas9 system, the protospacer-adjacent motif (PAM) is the critical element, located at the 3' end of the DNA target site, and dictating the search mechanism for the DNA target with Cas9 (1). Several studies have demonstrated that PAM is involved in the binding of the Cas9 to the target and the DSB (1). Target sequences without PAM do not induce DSB (4). Cas9 (formerly known as Cas5, Csn1, or Csx12) is the only enzyme within the Cas gene cluster that facilitates target DNA cleavage (1).

The CRISPR/Cas9, type 2 system is emerging as the sequencespecific nucleases of choice for genome engineering for three reasons (2):

1. Cas9 is guided by a single-guide RNA (gRNA) that is easily engineered. The gRNA targeting sequence consists of 20 nucleotides (nt), which is homologous to the DNA target site and can be ordered as a pair of oligonucleotides and rapidly cloned
2. The modular features of the CRISPR/Cas9 system and short 20nt length of the targeting gRNA makes these components advantageous in being able to target and cleave multiple target sequences simultaneously (multiplexing)
3. The CRISPR/Cas9 system enables efficiency and high specificity with minimal off-target effects of unwanted chromosomal translocations when well-designed gRNAs are used

The Cas9 requires precise homology between the gRNA and the targeted DNA sequence, but it does allow a few mismatches of base pairs in the target sequence when a DBS is generated (4). Depending on the number, position and distribution of mismatches, this could affect specificity and the desired application (1). These off-target effects and their long-term consequences are the current concerns with the CRISPR systems. Scientists are working on various methodologies, such as Cas9 nickase, to target single-strand breaks on opposite sides of the targeted DNA (1,4) – or choosing unique target sequences and optimising gRNA and Cas9 to minimise this phenomenon (5).

Discovery of the CRISPR/Cas9 system can be credited to two principal researchers: Jennifer Doudna, an award-winning scientist from the University of California and the Broad Institute, Massachusetts; and Emmanuelle Charpentier, a worldleading expert in the regulatory mechanisms underlying the processes of infection and immunity in bacterial pathogens – along with each of their teams. Both scientists have been shortlisted for the Nobel Prize (6).

In the laboratory, geneticists have been able to cut out HIV, correct sickle cell anaemia and alter cancer cells to make them more susceptible to chemotherapy (7). In the future, this technology may also make it possible to edit any human gene.

New Contender

Recently, Feng Zhang at the Broad Institute – who also worked on the CRISPR/Cas9 system – published his work in collaboration with various other institutions on the system using the enzyme Cpf1 instead of Cas9. It is predicted that this enzyme will help overcome some of the difficulties encountered by researchers as they develop CRISPR-related treatments for human diseases (8). Numerous companies have been working with the CRISPR/ Cas9 technology to create therapeutic treatments; however, none of these have yet reached clinical trials. While Cpf1 is still in the early research stage, Zhang believes that “there is little doubt that…there are additional systems with distinctive characteristics that await exploration and could further enhance genome editing and other areas of biotechnology, as well as shed light on the evolution of these defense systems.” (8)

Zhang offers the following perspective on why Cpf1 might be more advantageous than Cas9:

● Cpf1 uses only one strand of RNA compared to Cas9, which uses two strands to guide and reach its target gene. A single-strand system might be simpler, in addition to offering a cheaper design and an easier delivery process (enzyme-guide complex) into the cells (8,9)
● Cpf1 makes staggered double-strand cuts, or ‘sticky ends’, in the target DNA, whereas Cas9 cuts both DNA strands in the same location (‘blunt ends’) once it is delivered into the cell’s nucleus. Zhang et al writes that the staggered ends make it easier to insert a new gene after the old one is removed, because these unpaired nucleotides in that gene could only bond in the desired location. This could overcome the hurdles of Cas9, where scientists have said that replacing an old gene with a new one using Cas9 has proved more difficult than simply cutting out a gene (8,9)
● When Cpf1 hones in on a gene, it makes the cut-off to the side or further down the DNA strand. Zhang and his colleagues report that this feature could be “potentially useful” in preserving the target site for subsequent rounds of editing (8,9)
● In the Cpf1 protein, Zhang et al identified two enzymes from the Cpf1-family proteins: Acidaminococcus and Lachnospiraceae bacterium with efficient genome-editing activity in human cells. The Cpf1-family proteins are diverse in bacterial species, due to the T-rich PAMs of the Cpf1-family. This allows for applications in genome editing in organisms where all characterised mammalian genome-editing proteins require the presence of at least one G, so the T- and T/C-dependent PAMs of Cpf1-family proteins expand the targeting range of RNA-guided genome editing nucleases – one of the primary functions of CRISPR systems. For Cas9, only a small fraction of bacterial nucleases can function efficiently when heterologously expressed in mammalian cells (9)
Francisella Cpf1(FnCpf1) and crRNA alone are sufficient for mediating DNA targeting. Compare this to Cas9, which requires both crRNA and tracrRNA to mediate targeted DNA interference – thus making this feature a way to simplify the design and delivery of genome-editing tools (9)
Curing Single Genetic Disorders

It may be a while before gene editing can be applied to humans, and this step should be taken with extreme caution. Diseases involving more than one gene are so complex that implementation may require more progressive technology. Any mis-step may result in long-term adverse consequences.

If this can be achieved, medicine will change. Physicians will learn more efficient methods of diagnosing and treating diseases – and many now-common conditions could potentially be eliminated. Prevention would then be defined as editing a gene for a particular disease once it is diagnosed. Insurance providers will be more likely to pay for reimbursement of this new and highly specific preventive medicine, because once a disease is cured, the associated treatment expenses will fall over both the short term and long term. However, the one area that science will not be able to change any time in the extended future is the ageing process.

Weighing up the Pros and Cons

While advancing scientific technology is essential for curing diseases and improving crop yields, we must proceed with utmost care. Although scientists have the best intentions as they work diligently to advance medical research, there will inevitably be consequences to every action – both good and bad. Gene editing may allow people to live longer and with a better quality of life, but would this also create negative consequences, such as overpopulation? Or will Mother Nature find a way to maintain a balance with earthquakes, tornadoes and tsunamis – or with human-made disasters, such as terrorism and war?

Meanwhile, consumer technology entrepreneurs have simultaneously introduced devices such as radar guns that are employed in vehicle traffic control, microwave ovens and mobile phones – and are increasingly being used even in less affluent societies. While there are clear benefits to all these innovations, they also come with a downside, according to studies that associate their long-term use or exposure with the risk of cancer. Medical advances may offer the potential for a longer life, but both humans and Mother Nature have the ability to reverse these benefits.

Sometimes we become so enamoured with technology that the possibility of its unforeseen consequences to society are all but forgotten. Can the repercussions be anticipated, and can defensive measures be put in place to help avoid these? What has been learnt from our experience with genetically modified foods and high-tech devices that are supposed to make life easier? Future generations will likewise be impacted by consequences rooted in today’s technology. Life itself is a cascade or a domino effect; everything is related to and affected by everything else, much like systems biology.


1. Hsu P, Landers E and Zhang F, Leading edge review: Development and applications of CRISPR-Cas9 for genome engineering, Cell 157(6): pp1,262-1,278, June 2014. Visit:
2. Harrison M, Jenkins B and O'Connor-Giles KM, Review: A CRISPR view of development, Genes and Development 28: pp1,859-1,872, 2014. Visit:
3. Gaj T, Gersbach C and Barbas III C, ZFN, TALEN and CRISPR/Cas-based methods for genome engineering, Cell 31(7): pp397- 405, July 2013. Visit:
4. CRISPR in the lab: A practical guide. Visit:
5. Cho SW, Kim S and Kim Y, Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases, Genome Res 24(1): pp132-141, January 2014. Visit:
6. Steenhusen J, Nobel Prize predictions see honors for gene editing technology, Reuters, September 2015. Visit:
7. King MC, Charpentier E and Doudna J, April 2015. Visit:
8. Lash A, CRISPR update could make gene edits easier, discoverers say, September 2015. Visit:
9. Zetsche B et al, Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system, Cell 4:163(3): pp759-771, October 2015

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