CRISPR/Cas9 series: a compelling journey from a primitive bacterial defence system to an advanced human gene editing technology
DNA repair mechanisms as the key to engineering human genes by CRISPR/Cas9
Summary:
- In 2020, CRISPR/Cas9 technology achieved several breakthroughs that are revolutionizing the fields of food and medicine.
- CRISPR/Cas9 makes a break in DNA, triggering several cell repair mechanisms that researchers exploit to manipulate the genome of the organisms.
- The DNA repair system referred to as non-homologous end joining (NHEJ) is used to inactivate a gene, in order to investigate its function or to create models of disease.
- Homologous recombination-based DNA repair helps researchers to mend a defective gene or to insert a new one, as potential gene therapy for human diseases.
Despite the concerns about the global pandemic, 2020 will most likely also be remembered as the breakthrough year of CRISPR. Apart from the Nobel Prize in Chemistry awarded to Emmanuelle Charpentier and Jennifer Doudna, CRISPR/Cas9 technology has come under the spotlight for other exciting reasons.
In April 2020, scientists at University of California, Davis (UC Davis) breeded Cosmo, the first CRISPR genetically engineered bull calf [1]. In December, the U.S. Department of Agriculture (USDA), following the Food and Drug Administration (FDA) decision, regulated the use of CRISPR/Cas9 as a new method to obtain genetically engineered crops [2-3]. Last summer, CRISPR/Cas9 was successfully used to produce healthy blood cells for treating a woman affected by sickle cell anemia [4]. Furthermore, a few months earlier, results from a clinical trial at University of Pennsylvania demonstrated that use of human T lymphocytes engineered by CRISPR/Cas9 could be feasible for cancer therapy [5].
In our previous article, we presented how CRISPR/Cas9 technology was derived from the adaptive immune system of bacteria and how it works. CRISPR/Cas9 is a complex formed by a protein (Cas9) and a guide RNA (gRNA) that is able to recognize a specific DNA sequence and create a break within it. In this article, we cover why being able to cut DNA precisely is crucial for the researchers and how this started the revolution we are witnessing in the genetic engineering field.
DNA breaks are a natural phenomenon in many organisms. For instance, physiological DNA breaks can occur during cellular division to facilitate DNA exchange between the pairs of chromosomes. Moreover, external stimuli, such as UV radiation and chemical agents, can cause damage to the DNA of cells, which normally consists of one or more breaks of the double helix. In order to survive, it is imperative that cells repair DNA lesions whenever they occur. Analogously, the Cas9 cut results in a targeted DNA break to a specific sequence that scientists want to edit by taking advantage of the cell repair mechanisms.
In fact, in higher organisms, including humans, DNA breaks can be recovered by several sophisticated repair mechanisms [6-7]. The most frequent of these mechanisms is referred to as non-homologous end-joining (NHEJ). As a result of damage to DNA sequences, this repair system inserts or deletes small pieces of DNA at the breakage site to reconstruct the damaged sequence. However, this mechanism is highly error prone and following random insertion, as well as deletions, of small DNA pieces may result in dramatic changes of the original DNA sequence [8].
Researchers take advantage of the errors occurring during DNA repair in order to abrogate the expression of a specific gene. Indeed, random mutations often result in a non-functioning gene, then in a non-functioning protein. Abrogation of genes is very helpful to investigate the biological effects of their removal, to study a signaling pathway or to create disease models to assay new therapeutic approaches. Nevertheless, the randomness of the changes introduced by NHEJ allows for little control in gene editing by researchers. In particular, this system can not be used to correct a gene that causes a disease, by re-establishing its function, or to give new advantageous features to the manipulated cells [9].
To overcome such limitations, scientists exploit a second, less frequent repair system based on homologous recombination. Homologous recombination is a molecular process providing high-fidelity repair in which genetic information is exchanged between two similar molecules of DNA. When a break in genomic DNA occurs, an intact identical molecule of DNA is used by the cells to substitute the damaged ones.
Using this principle, researchers are able to introduce a new sequence into a host cell genome in a rather simple way:
- The Cas9/gRNA complex is introduced into cell nuclei to cut a specific sequence of genomic DNA;
- Researchers also introduce a molecule of DNA as homologous template, containing the modifications they want to introduce;
- Cas9-mediated break is repaired by homologous recombination of the introduced DNA template; in other words, cells are tricked into repairing their own genome with the chosen modifications [9-10].
In conclusion, CRISPR/Cas9 represents an intriguing example of how scientists invented a genome editing tool by combining a small prokaryotic defence mechanism and the eukaryotic DNA repair mechanisms. This opened the door to the proposal of next generation therapeutic strategies for the treatment of genetic diseases, cancer and many others. However, despite the fascinating potential of CRISPR/Cas9, researchers faced several challenges in the application of this technology.
In the next article of this series, we will address the major technical drawbacks of the CRISPR/Cas9 toolbox, such as off-target events, and we will discover how the scientists have overcome these issues and improved the system for human application.
References:
- https://ucdavis.app.box.com/s/cpipr5wwnrdr69k7s46l81kbtmzycfg7/file/694452573988
- https://www.aphis.usda.gov/brs/pdf/aphis-2020-0079.pdf
- https://www.fda.gov/media/74614/download
- https://www.npr.org/sections/health-shots/2020/12/15/944184405/1st-patients-to-get-crispr-gene-editing-treatment-continue-to-thrive
- https://science.sciencemag.org/content/367/6481/eaba7365
- Liang F, Han M, Romanienko P.J., & Jasin M. Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proceedings of the National Academy of Sciences, 95(9), 5172-5177 (1998)
- Ranjha, L., Howard, S. M., and Cejka, P. (2018). Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes. Chromosoma 127, 187–214. doi: 10.1007/s00412-017-0658-1
- Bothmer, A. et al. Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus. Nat. Commun. 8, 13905 (2017).
- Sonoda E, Hochegger H, Saberi A, Taniguchi Y, Takeda S. Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair (Amst) 2006; 5: 1021–1029.
- Horii, Takuro, and Izuho Hatada. “Challenges to increasing targeting efficiency in genome engineering.” The Journal of reproduction and development vol. 62,1 (2016): 7-9. doi:10.1262/jrd.2015-151