Accuracy in gene editing methods is highly sought after to facilitate accurate research, especially in modeling disease. Gene editing is a subset of the umbrella term of genetic engineering, it is a process wherein sections of target DNA are modified either by point mutation such as insertion, deletion, modification, or replacement.
Gene Editing. Image Credit: Natali _ Mis/Shutterstock.com
The most commonly used are modified nucleases, enzymes that cleave the nucleotide chains that constitute the backbone of DNA, such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), meganucleases, and CRISPR/Cas9. The most popular and new method of these is CRISPR, also known as clustered regularly interspaced short palindromic repeats. Medical news surrounds the progress made by the use of CRISPR/Cas9 gene editing, especially in fields where research has been long stagnant, such as chronic disease or neurodegenerative disorders.
The discovery and success of CRISPR were supported by its predecessors. The history of gene editing shows an interesting chain of progress that can predict its future trajectory, currently, rapid advancements are unearthed daily. Here, the different methods will be explained, new studies will be discussed, and future possibilities explored.
Early Discoveries Paved Way for Future Success
Genetic engineering first emerged in the form of recombinant DNA, where genes from a certain organism are transplanted into another. The Berg lab inserted SV40 bacteriophage genes into E. coli bacterial genome in 1972 and used it to extensively study bacterial antibiotic resistance. This does not constitute as gene editing as it integrates constructs into a host genome.
In 1985, zinc finger domains are protein structures that were discovered from studies using Xenopus (frog) oocytes observing the interaction with a Xenopus transcription factor (TFIIIA) with 5S rRNA (ribosomal RNA). Later studies clarified the 3D structure and ability to selectively switch genes “on'' or “off”.
Novel site-specific endonucleases, enzymes that cleave nucleotides at points that aren’t at either end, were generated from 1996 by combining two different zinc finger proteins to the cleavage domain of FokI endonuclease. This marked the creation of zinc finger nucleases (ZFNs) that could “cut” DNA near a pre-determined target.
TALENs were discovered in the 2010s, relatively recently in comparison. TALE proteins are secreted by Xanthomonas bacteria when infecting plants. Similar to zinc finger domains, TALE proteins were associated with FokI endonucleases to target specific gene sections.
Meganucleases constitute a family of naturally occurring, rare-cutting endonucleases observed recently in the early 2010s. I-SceI is used for genome editing, naturally, it functions to stimulate homologous recombination, where genetic information is exchanged between two similar or identical molecules, by stimulating a specific double-stranded break within the genome. Making use of this is promising for gene editing, however, is impractical for therapeutic situations due to the need to introduce a known cleavage site to the region of interest.
CRISPR/Cas9: Cutting Edge Technology
One of the most popular methods of gene editing currently is the use of CRISPR/Cas9. While the term “CRISPR” was coined in 2000, development and discovery began in 1987 where 29 nucleotide repeats interrupted by shorter “spacer” sequences were noted in E. coli, reports of similar sequences in other microorganisms followed. It was in 2008 that DNA targets were uncovered. Spacer sequences could be transcribed into CRISPR RNA (crRNA) which can act as small guide RNAs (sgRNA) which guide Cas endonucleases to generate double-strand breaks.
Initially, the CRISPR/Cas interaction facilitated the immunological response in prokaryotes spacer sequences incorporate foreign DNA acquired via prior infection and used to protect from subsequent infection. Interestingly, Cas genes clustered closely to these spacer genes.
Furthermore, in 2012, in-vivo targeted gene editing was accomplished with specificity by the use of sgRNA. CRISPR use in human cells was first accomplished in 2013 when targeted genome cleavage in mouse and human cells was demonstrated. The same team also showed that the CRISPR/Cas9 system can be programmed to target multiple gene loci and drive homology-directed repair. Within eukaryotes, Cas9 is the favored nuclease due to its availability across different organisms.
|
Different DNA Repair Techniques
|
|
Homology Directed Repair
|
Generally, when a homologous sequence is available within the nucleus when there is a double-stranded break.
e.g. homologous recombination, single-strand annealing, and breakage-induced replication
|
|
Non-Homologous End-Joining
|
Fixes double-stranded breaks without the need for a homologous template.
|
CRISPR/Cas-9 Gene Editing Clinically Improve Sickle Cell Disease
On December 5th, 2020, the New England Journal of Medicine published an article wherein successful treatment and improvement of two potentially life-threatening diseases (sickle cell disease and transfusion-dependent beta-thalassemia) within two patients with either disease was reported. This was done by electroporation of CRISPR-Cas9 into CD34+ hematopoietic and progenitor cells from healthy donors which target the BCL11A erythroid-specific enhancer to improve the production of fetal hemoglobin (which has a greater affinity for oxygen) among other functions.
These cells were transfused after a process of myeloablation, where the patients are subjected to full body irradiation to destroy bone marrow/stem cells and replaced by transfusion of CRISPR/Cas9 altered BCL11A promoted cells. The patients were monitored for a year; the released article details the exciting improvement seen within this novel therapy.
Anti-CRISPRs Sharpen CRISPR Precision
While CRISPR is a generally precise tool, it is not infallible. It is vastly more accurate than preceding technology but is still subject to off-target effects that may influence the action of the intended target.
This precision problem was much higher with the use of ZFN and TALENs comparative to CRISPR, however, when working with precise gene editing for therapeutic purposes, the highest accuracy possible is necessary. This is where Anti-CRISPR (Acr) proteins come in, Acr protein AcrIIA4 forms a complex with Cas9, and if delivered at the appropriate time, allows for on target gene-editing whilst limiting off-target effects.
Conclusion
There are a variety of gene editing tools available, the most efficient and accurate is the CRISPR/Cas9 system. The favored attributes include specificity and accuracy, meaning there are fewer off-target effects. While CRISPR is the desired tool of choice today, future developments may work to hone its ability or possibly discover a new method entirely.
Sources:
- Nemudryi AA, Valetdinova KR, Medvedev SP, Zakian SM. TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery. Acta Naturae. 2014;6(3):19-40.
- Klug, A. (2010). The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation. Quarterly Reviews of Biophysics, 43(1), 1-21. doi:10.1017/S0033583510000089
- Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Y G Kim, J Cha, S Chandrasegaran. Proceedings of the National Academy of Sciences Feb 1996, 93 (3) 1156-1160; DOI: 10.1073/pnas.93.3.1156
- Xanthomonas AvrBs3 Family-Type III Effectors: Discovery and Function. Jens Boch and Ulla Bonas. Annual Review of Phytopathology 2010 48:1, 419-436
- Joung JK, Sander JD. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol. 2013;14(1):49-55. doi:10.1038/nrm3486
- Silva G, Poirot L, Galetto R, et al. Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr Gene Ther. 2011;11(1):11-27. doi:10.2174/156652311794520111
- Shin J, Jiang F, Liu JJ, et al. Disabling Cas9 by an anti-CRISPR DNA mimic. Sci Adv. 2017;3(7):e1701620. Published 2017 Jul 12. doi:10.1126/sciadv.1701620
- Gupta, D., Bhattacharjee, O., Mandal, D., Sen, M.K., Dey, D., Dasgupta, A., Kazi, T.A., Gupta, R., Sinharoy, S., Acharya, K., Chattopadhyay, D., Ravichandiran, V., Roy, S. and Ghosh, D. (2019). CRISPR-Cas9 system: A new-fangled dawn in gene editing. Life Sciences, 232, p.116636.
- Broad Institute (2018). CRISPR Timeline. [online] Broad Institute. Available at: https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/crispr-timeline.
- 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., Ho, T.W., Kattamis, A., Kernytsky, A., Lekstrom-Himes, J., Li, A.M., Locatelli, F., Mapara, M.Y., de Montalembert, M., Rondelli, D. and Sharma, A. (2020). CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. New England Journal of Medicine.
Further Reading