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A Guide To Manipulating Your DNA

More and more gene editing techniques are emerging in the world of biotechnology, allowing advancements in medicine, pharmaceuticals and agriculture. In biomedical lab research, gene manipulation allows researchers to analyze gene function within the context of a cell and turn on or off gene products to observe the effects. The most impressive possibilities though are the ability to add new functions to the cell or even reverse genetic diseases. This article explains the biochemical background of the most popular gene editing methods, such as RNA interference, Zinc finger nucleases, TALENS and CRISPR.

RNA Interference

Back in 1998, Fire and Mello, observed that Caenorhabditis elegans (C. elegans) worms that carried a specific mutation, displayed a twitching behavior. The two scientists examined if introducing various forms of RNA sequences that matched an RNA responsible for a muscle protein in the worm, would block production of that protein and result in twitching of the worm. The first molecule that they tested was a single-stranded RNA that was complementary to the target RNA. To their surprise, this molecule did not cause any twitching. However, when they introduced a double stranded RNA, the worms twitched, signaling that the targeted gene was silenced. This experiment led to the finding that administration of double stranded RNA into the cell causes destruction of a matched RNA sequence in the organism’s cells. This process is known as RNA interference (RNAi) (Fire et al., 1998). It is thought that this is a natural mechanism that evolved as a defense system against viral RNA. Once the foreign double stranded RNA enters the cell, an enzyme, called Dicer, processes it into a small interfering RNA (siRNA). Consequently, the RISK complex loads one siRNA band. This complex now targets a specific mRNA sequence, hybridizes to it through complementarity and cuts it into fragments. In biotechnology, a specific RNA molecule is introduced into a cell with various methods, including using a plasmid or through electroporation, and the process of RNAi leads to the silencing of the gene of interest (Agrawal et al., 2003).

Figure 1 - C. elegans Twitching (Rohl, 2006)

Zinc Finger Nucleases and TALENs

Some others widely used gene editing tools are Zinc Finger Nucleases (ZFNs). ZFNs are synthetic proteins that are used to insert or delete parts of the DNA in specific sites in the genomes of cells. It was found that transcription factors normally include zinc finger motifs. Each motif recognizes three base pairs (Urnov, Rebar, Holmes, Zhang, & Gregory, 2010). ZFNs are a combination of a zinc finger domain fused with an enzyme. A different combination of zinc fingers is used each time to target a specific sequence. These zinc fingers are linked to the nuclease domain of a restriction enzyme called FokI. ZFNs are usually dimerized (two ZFN subunits are combined) to recognize a target sequence and cleave it in the desired place (Cathomen & Keith Joung, 2008).

Transcription activator-like effector nucleases (TALENs) is another editing tool with a similar function to ZFNs. TALEs are bacterial transcription factors that are secreted during plant infection. They are a combination of many TALE repeats. Each repeat recognizes and binds to a single base. Similarly to ZFNs, a specific combination of TALEs is fused with the nuclease domain of a FokI enzyme to make a TALEN. In order to edit a piece of DNA, a dimerized TALENs molecule is used to cleave out a specific sequence (Bhardwaj & Nain, 2021).

Figure 2: Genome Editing in the Lab (Mah et al.)

The Nobel Prize Awarded To CRISPR

Clustered regularly interspaced small palindromic repeats (CRISPR) are some of the most interesting topics in biochemistry, with news and updates filling up our newsfeed every day. The development of this technique by Jennifer Doudna and Emmanuelle Charpentier was awarded with the Nobel prize in Chemistry in 2020. CRISPR is a bacterial and archaeal defense system that protects the host organism from phages, viruses and plasmids (Barrangou et al., 2007). It consists of the CRISPR associated (Cas) genes, encoding for the Cas proteins, and the CRISPR array. The latter contains spacer sequences, inserted into the bacterial genome by a virus, and short palindromic repeats. Transcription of the CRISPR array generates crRNA; short RNA sequences consisting of the transcribed spacer sequence and the palindromic repeat. The crRNAs along with a transactivating crRNA (tracrRNA) are obtained by CRISPR effector nuclease enzymes (Cas enzymes). When the same virus attacks the bacterium, the Cas enzymes recognize its genome through complementarity and destroy it. They identify the Protospacer Adjacent Motif (PAM) and the protospacer, which is the original spacer sequence inserted by the virus. PAM is a short nucleotide region next to the protospacer, specific for each Cas nuclease (Bolotin, Quinquis, Sorokin, & Ehrlich, 2005).

There are several types of CRISPR systems differentiated by their architecture and the cas genes that they encode. Type II systems express the Cas9 protein, which contains a RuvC nuclease domain and an HNH nuclease domain. It identifies the PAM sequence 5’-NGG-3’ at the 3’end of the protospacer. Once Cas9 locates these two elements, it forms an R-loop with the foreign DNA and cleaves it at specific sites (Makarova et al., 2011).

CRISPR/Cas9 functions as a programmable nuclease for gene editing. Cas9 can be associated with a single guide RNA (sgRNA), that contains the crRNA and the tracrRNA scaffold. This sgRNA targets a particular sequence that is cleaved by the Cas9 nucleases.

Figure 3: How CRISPR works (Falconer, 2023)

Current Limitations

Gene editing techniques are evolving day by day. Interestingly, a CRISPR-like programmable endonuclease called Fanzor was recently found in eukaryotes (Saito et al., 2023). Although all these techniques show great potential in gene editing, their use in human organisms is still impossible. Some issues include the off-target effects, administration issue and toxicity events. However, gene editing seems to be the future in biomedicine, giving the opportunity to scientists to target disorders (especially genetic ones) to their core, and correct genetic malfunctions.


RNAi, ZFNs, TALENs and CRISPR are the most well-researched gene editing techniques at the moment. Along with the help of bioinformatics, they allow scientists to target specific sequences and introduce edits such as insertions or deletions. Gene editing seems to offer a revolutionary approach to research, medicine and agriculture and promises great achievements in the future.

Bibliographical References

Agrawal, N., Dasaradhi, P. V., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., & Mukherjee, S. K. (2003). RNA interference: Biology, mechanism, and applications. Microbiology and Molecular Biology Reviews, 67(4), 657–685. doi:10.1128/mmbr.67.4.657-685.2003

Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., … Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science, 315(5819), 1709–1712. doi:10.1126/science.1138140

Bhardwaj, A., & Nain, V. (2021). Talens—an indispensable tool in the era of CRISPR: A mini review. Journal of Genetic Engineering and Biotechnology, 19(1). doi:10.1186/s43141-021-00225-z

Bolotin, A., Quinquis, B., Sorokin, A., & Ehrlich, S. D. (2005). Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology, 151(8), 2551–2561. doi:10.1099/mic.0.28048-0

Cathomen, T., & Keith Joung, J. (2008). Zinc-finger nucleases: The next generation emerges. Molecular Therapy, 16(7), 1200–1207. doi:10.1038/mt.2008.114

Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature, 391(6669), 806–811. doi:10.1038/35888

Makarova, K. S., Haft, D. H., Barrangou, R., Brouns, S. J., Charpentier, E., Horvath, P., … Koonin, E. V. (2011). Evolution and classification of the CRISPR–Cas Systems. Nature Reviews Microbiology, 9(6), 467–477. doi:10.1038/nrmicro2577

Saito, M., Xu, P., Faure, G., Maguire, S., Kannan, S., Altae-Tran, H., … Zhang, F. (2023). Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature. doi:10.1038/s41586-023-06356-2

Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., & Gregory, P. D. (2010). Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics, 11(9), 636–646. doi:10.1038/nrg2842

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Matina Laskou

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