DNA is usually depicted as a book in which the information contained within - given as a combination of triplets of A, T, G or C – provides the instruction for the development and the function of a living organism. For a long while, DNA has been deeply studied but the information contained within was still considered inaccessible to human manipulation. However, for the past 20-years, this is not true anymore as the newest discoveries in molecular biology have granted us the tools to interact with the DNA like never before.
Gene editing in a nutshell
Gene editing (or genetic engineering) entails the ability to manipulate the genetic information contained within the DNA by using molecular biology tools also known as “molecular scissors”. As the name suggests, molecular scissors are DNA nucleases that can be programmed to cut the DNA at the desired location. The cut usually occurs on both strands of the DNA double helix and therefore it is also defined as Double-Strand-Break(DSB). Gene editing takes advantage of the endogenous DNA repair mechanisms which are triggered when a DSB is detected by the cells. This complex cascade of events is also known as DNA Damage Response(DDR). A DSB damage can be generally repaired via two main mechanisms: the Non-Homologous End-Joining (NHEJ) and the Homology-Directed Repair (HDR). The NHEJ is regarded as an error-prone mechanism meaning that when the DSB is repaired some errors may occur because of which DNA bases may be mistakenly added (Insertions) or they can be lost (Deletions), shortened to indels. Addition or deletion of bases within the portion of a gene codifying for the respective mRNA – defined also as coding sequence – may alter the so-called reading frame, meaning how the triplets are read by the transcriptional machinery. This can result in mutations generating a premature stop codon (nonsense mutation) or the protein produced contains the wrong amino acid (missense mutation). Gene editing relies particularly on generating nonsense mutations to inactivate a gene, this is also defined as Knock-Out (KO). On the other hand, HDR is an error-free mechanism and the DSB repair is guided by the presence of a homology sequence to identify a DNA portion to be used as a blueprint to repair the DSB without introducing any indel. Usually, the “natural blueprint” is contained within the homologous chromosome. However, for gene editing applications it is possible to design an “artificial blueprint” that is made of the same homology arms of the homologous chromosome but in between the two homology arms (left and right), it can be inserted a desired DNA sequence. This way it is possible to integrate an exogenous DNA sequence where the cut has been generated by the molecular scissors. This practice is also known as targeted integration or Knock-In (KI) .
Molecular scissors and CRISPR/Cas
As we saw, gene editing relies on the natural DNA repair mechanisms already present in the cells to repair DNA damage. It is possible to trigger these mechanisms by using these novel tools named molecular scissors.
Although they have been around for almost 20 years, only in the past 8 years or so we have started hearing about gene editing more often.
As you may have already guessed, we are speaking about Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). In nature, CRISPR is a bacteria defense system that is activated to protect the bacteria from the invasion of exogenous infections such as bacteriophages and plasmid DNA (Fig.1).
Once that the infection is detected, the CRISPR locus containing a set of DNA nucleases – known as Casproteins – is synthetized along with an array of so-called guide RNAs (gRNAs). The guide RNA possesses a portion of variable length called spacer which is complementary to the intended target, called protospacer. Notably, the target besides possessing a matching protospacer must also have immediately next to it the so-called Protospacer Adjacent Motif (PAM) for Cas:gRNA complex to cut the DNA. By associating with the Cas protein, the gRNA guides it toward its target, the infecting DNA agent. Each bacteria species has its respective CRISPR system. In 2012, the laboratory of Jennifer Doudna and Emanuelle Charpentier demonstrated that it was possible to repurpose the CRISPR system of the Streptococcus pyogenes for gene editing. Particularly, the Streptococcus pyogenes main Cas protein is the Cas9 nucleases (required PAM is NGG, where N stands for any nucleotide). They proved that by simply changing the spacer sequence – 20 nucleotides - of the gRNA it was possible to “reprogram” the Cas9:sgRNA complex toward any possible target in the genome.
To understand the importance of this we must go back to two of the previous molecular scissors: Zinc-Finger Nucleases (ZFNs) and Transcriptional activator-like Effector Nucleases (TALENs) (Fig.2). They constitute the two major molecular scissors before CRISPR/Cas. Although different, they are both based on a similar architecture: they possess a DNA binding domain fused to a Fok-I DNA cleavage domain. To programthese scissors to cut a defined targeted some key regions of the DNA binding domain– also called modules - must be redesigned. This entails a re-engineering of the protein itself which takes time, money and great knowledge of how such modules have to be modified to interact with the desired target.
On the other hand, CRISPR/Cas is a binary system where the cut of the DNA is performed by the Cas9 nuclease and the target identification is done by the sgRNA. To target the Cas9 nuclease, it is necessary to only change the spacer sequence contained in the sgRNA. This practice is much simpler than modifying the modules of ZFNs or TALENs. The design and generation of a new sgRNA can take 3 days if it is self-produced in the lab or it can be done just in one click if ordered from a biotech company. Conversely designing new modules can take weeks to generate and a lot of hands-on work and money.
So, it is not hard to understand why a lot of labs immediately started adopting CRISPR and using it for different purposes both in basic and applied research. Such applications were covered in the past by ZFNs and TALENs as well but to required much more time, money and expertise to employ them. Conversely, thanks to its ease of use and low cost an increasing number of laboratories from all over the world have started using CRISPR almost daily.
There has never been a better time to work in Gene Editing
CRISPR/Cas technology has primed a new era for the Gene Editing field and Molecular Biology in General, so that Jennifer Doudna and Emmanuelle Charpentier were awarded the Nobel Prize for Chemistry in 2021. CRISPR/Cas has become one of the primary tools in basic research and applied research to investigate new biological functions along with developing more accurate disease models . Even pharma companies have started using it as a tool to screen for validating novel targets to help the process of drug design. Ultimately, CRISPR/Cas has also entered the precision medicine field and it has been successfully used in the last 2-years in the first human clinical trials . While this on the one hand proves the immense potential of this technology – testified also by the growing number of CRISPR-based biotechs - it also shows that in the years to come there will be a growing need for people with understanding and expertise in the use of CRISPR/Cas and gene-editing technology. Therefore, if you are among the ones who are enthusiastic about this field there has never been a better time to dig much more into it and develop a unique set of skills that will be highly demanded in the next future and will help to push forward the boundaries of biology and precision medicine.
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2. Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J.A.; Charpentier, E. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science (80-. ). 2012, 337, 816 LP – 821.
3. Li, H.; Yang, Y.; Hong, W.; Huang, M.; Wu, M.; Zhao, X. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduct. Target. Ther. 2020, 5, 1.
4. Frangoul, H.; Altshuler, D.; Cappellini, M.D.; Chen, Y.-S.; Domm, J.; Eustace, B.K.; Foell, J.; de la Fuente, J.; Grupp, S.; Handgretinger, R.; et al. CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. N. Engl. J. Med. 2020, 384, 252–260.
Meet the Author
• Ph.D student at the Center for Transfusion Medicine and Gene
Therapy, Freiburg (Germany)
• ASGCT Excellence in Research Award 2021
• Freelance scientific writer
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