Ario Azarhoush | SQ Vol. 20 Research Features (2022-2023)
Living organisms often suffer from genetic disorders: diseases caused by genetic mutations, or changes from an organism’s normal DNA sequence. These alterations can either occur during the early stages of development—causing the organism to be born with the disorder—or throughout an organism’s lifetime.1 For generations, scientists have strived to develop a reliable genome editing technology with the potential to treat genetic disorders and diseases. With genome editing technologies, many genetic disorders that were once thought to be incurable, such as Huntington’s disease, Parkinson’s disease, cystic fibrosis, and sickle cell anemia, have the potential of being treated.2 In the past decade, CRISPR-Cas9 has emerged as one of the most prominent advances in modern biotechnology with the potential to accomplish just that. Within this rapidly expanding field, UC San Diego has emerged as a pioneer in CRISPR-Cas9 research. Biologists from UC San Diego have even developed the world’s first CRISPR-Cas9 approach to control genetic inheritance in mammals.3
As an undergraduate majoring in General Biology, Andres Sandoval was motivated to pursue research at UC San Diego from a desire to strengthen public health initiatives through applications in t agricultural sciences. In agriculture, CRISPR-Cas9 can be used to remove undesirable genes and add desirable genes to grow plants with ideal characteristics. For instance, a scientist could add certain genes to crops that will make them grow larger or faster, or genes that make the crops more resistant to climate change.4 Conversely, scientists can also remove genes that may make plants more susceptible to disease, or genes that make the plots grow suboptimally.5 The promise of this new technology has led researchers to dedicate their work to investigating the molecular mechanisms that can help people to refinine editing efficiency. At UC San Diego, Andres Sandoval worked with Professor Lisa McDonnell to test the occurrence and efficiency of gene editing by testing various homology arm configurations around the DNA cut site.
However, understanding the molecular mechanisms of CRISPR-Cas9 starts with learning the central dogma, or basic mechanics, of molecular biology. The central dogma states that genetic information flows from DNA to RNA to protein. Essentially, DNA is a long molecule that exists within all living organisms and stores genetic information needed to develop and function. The basic structural units which are strung together to form DNA are known as nucleotides. There are four nucleotides which make up DNA: adenosine (A), thymine (T), guanine (G), and cytosine (C). These nucleotides join together in a double-stranded configuration and form base pairs. These base pairs are arranged into sequences of three nucleotides called codons; each codon encodes a specific amino acid—the basic unit of a protein. In humans, a series of codons encodes a gene, or the DNA sequence for an entire protein. However, in order to create a protein, cells require an intermediate molecule to facilitate the translation from a DNA sequence. DNA strands serve as a template for RNA, or ribonucleic acid. Like DNA, RNA is also made up of nucleotides and codons. To synthesize RNA molecules, specific proteins copy a particular DNA sequence through a process called transcription. These RNA transcripts act as messengers, carrying the codons from DNA to other cellular machinery that read the transcript to build proteins through a process called translation. These proteins then carry out many different functions essential to sustaining the life of a cell. These processes include: maintaining cell and tissue structure, facilitating essential chemical reactions, and protecting the organism against diseases.
Despite encoding for essential cellular processes, DNA can be damaged through mechanistic, chemical, and environmental factors. In its most extreme form, damage can occur as double-stranded breaks, or breaks in both of the paired DNA strands. Double-stranded breaks in important genes can cause nucleotides along the broken ends to be eroded away, or lost, preventing the synthesis of crucial regulatory proteins which, if not repaired, can result in genetic degradation and ultimately cell death. If a double-stranded break occurs in cells during the early stages of embryonic or cell development, it can lead to the onset of genetic disease, and in the worst case scenario, death of the organism.
Luckily, the genome possesses mechanisms that are capable of repairing these breaks to prevent genetic degradation. Double-stranded breaks can be repaired through two potential pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ repairs DNA by directly ligating, or joining, the broken ends together.6 By directly ligating the broken ends together, sequences that may have been lost in the break cannot be recovered, and therefore NHEJ is considered the more “error-prone” double-stranded break repair mechanism. In contrast, HDR uses a homologous or functionally identical gene as a template to fill in sequences lost in the double-stranded break.7 Since HDR uses homologous DNA as a template for genetic repair, it is more accurate and precise in comparison to NHEJ, making it the less “error prone” repair mechanism. Within cells, NHEJ and HDR mechanisms naturally compete with one another to repair the break. Thus, it is generally up to random chance whether NHEJ or HDR is chosen.
By harnessing naturally occurring double-stranded break repair mechanisms, CRISPR systems enable scientists and medical researchers to add, remove, or alter parts of the DNA sequence. These intentional alterations are also known as mutations. Mutating the DNA sequence changes the instructions that RNA molecules copy, and therefore may alter the final protein product. Some mutations may not cause any changes in the final protein created, known as silent mutations. However, others can drastically reduce the ability of the protein to function, or lead to the production of a completely different protein altogether. On a larger scale, these changes can lead to changes in or the prevention of the expression of particular traits.
In order to introduce mutations, CRISPR-Cas9 systems need to start by introducing a double-stranded break in a desired location of the genome. This break is facilitated by a Cas9 nuclease. Nucleases are proteins best described as a pair of molecular scissors that excise, or cut, both complementary strands of a DNA sequence8. To locate this region of interest, the CRISPR-Cas9 system also utilizes a special type of RNA called a guide RNA (gRNA). Researchers design the gRNA to code for a particular sequence of interest. Just as the name suggests, the gRNA is designed to recognize a desired DNA sequence, and then guide the Cas9 nuclease to the sequence so that the DNA sequence can be cut. Finally, researchers induce DNA repaired at the region of interest to modify a select characteristic.
To introduce new genetic sequences, the CRISPR-Cas9 system uses HDR to incorporate intentional mutations to the repaired DNA. Mutations can be introduced by providing a designed DNA template, known as a donor DNA, for HDR to copy when repairing the break. Recent studies have attempted to manipulate the structure of the donor DNA to optimize the efficiency and accuracy of HDR incorporating the desired mutations during repair of the double-stranded break. One such structure is the presence and configuration of homology arms on the donor DNA template. Homology arms are designed to base pair to regions that surround or flank the DNA cut site, to help the CRISPR-Cas9 complex accurately locate the cut site.
Currently, the ideal configuration of homology arms that flank the cut site to optimize implementing a mutation utilizing HDR is not well understood. In an effort to dissect the mechanisms to increase the efficiency of CRISPR Cas9, Sandoval sought to test whether HDR templates are required to flank both upstream or downstream of the DNA cut site. To identify potential factors, Sandoval used a circular form of DNA, known as a plasmid, that was designed to code for a particular gRNA and a Cas9. This plasmid, referred to as pML104, would eventually be inserted into a particular yeast colony. This process is known as transformation, and allows the plasmid to retain the yeast genes while utilizing yeast as a host to complete the CRISPR process. Inserting a plasmid into a yeast cell is similar to how a DVD is used in a DVD player to play a movie. The plasmid retains the genetic information for CRISPR components, while the yeast cells act as the host cell for the CRISPR process to occur within. Yeast are often a popular host for CRISPR studies due to their efficient use of HDR, so researchers can precisely edit their genome. Sandoval edited the yeast genomes by taking advantage of a gene in yeast called ADE2, a gene that is crucial for yeast colonies to stay alive. The color of the colony serves as a visual marker for alterations to this gene: a functional ADE2 gene will cause the yeast to appear white, but a nonfunctional ADE2 gene will cause the colony to produce a red pigment.
Using pML104, Sandoval designed a donor DNA with silent mutations. Silent mutations were introduced by changing the last nucleotide, or the wobble position, of a few codons in the ADE2 gene, altering the overall DNA sequence. This changed the nucleotide sequence of the codons, but did not alter the amino acids they encoded. Altering the wobble position allowed Sandoval to introduce silent mutations, or mutations that do not change the protein structure from the ADE2 gene, allowing the edited yeast to still produce a white pigment. A total of seven silent mutations were implemented into the HDR template.
From here, Sandoval designed three experimental groups. One group, called HDR D, would have these mutations only upstream of the DNA cut site, while having homology arms only downstream of the cut site. The second group, HDR C, would have these mutations only downstream of the site where DNA was cut by Cas9, while having homology arms only upstream of this site. The final group, HDR B, would have these mutations both upstream and downstream of the DNA cut site with homology arms flanking both upstream and downstream after the mutated sequences. This experimental design allowed for HDR to be the prioritized mechanism of repair rather than NHEJ, though some NHEJ would still occur.
Once the transformed yeast colonies had grown, Sandoval returned to his bench to find his yeast culture plates littered with hundreds of red and white spots. Colonies with the strongest white growth were selected for genome sequencing to determine whether any of these colonies were successfully edited with the CRISPR-Cas9 system. Sequencing data allowed Sandoval to identify that only HDR B was able to successfully incorporate partial or entire HDR mutations. These results suggest that the HDR mutations using CRISPR-Cas9 is optimal when homology arms flank both upstream and downstream of the cut site.
New genome editing technologies such as CRISPR must constantly be improved upon to increase their viability in being able to accurately edit the genome. When examining the conclusions of this experiment on a macroscopic level, the results of Sandoval’s research serve as a method for improving the efficiency of HDR gene editing with the CRISPR-Cas9 process. Accordingly, Andres Sandoval hopes that his research “will indirectly benefit the future of humankind.” He believes that his research with Professor McDonnell has the potential to increase the efficiency of CRISPR-Cas9, a technology that Sandoval sees as having “the potential to benefit so many lives in so many ways.” More recent applications of CRISPR in agriculture have already shown the promise of the novel technology to bolster public health.9 Ultimately, contributions like Andres Sandoval’s research will allow CRISPR to evolve into a tool that may safely and reliably be utilized in humans to eliminate fatal genetic disorders and improve quality of life on a monumental scale.
References
- Genes, Behavior, and the Social Environment: Moving Beyond the Nature/Nurture Debate. 2006. National Academies Press (US).
- Liu, W., Li, L., Jiang, J., Wu, M., & Lin, P. 2021. Applications and challenges of CRISPR-cas gene-editing to disease treatment in clinics. Precision Clinical Medicine, 4(3), 179–191. 10.1093/pcmedi/pbab014
- Aguilera, M. (2019, January 23). UC San Diego Researchers First to Use CRISPR/Cas9 to Control Genetic Inheritance in Mice. UC San Diego School of Biological Sciences.
- Zaidi, S. S.-A., Mahas, A., Vanderschuren, H., & Mahfouz, M. M. 2020. Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants. Genome Biology, 21(1). https://doi.org/10.1186/s13059-020-02204-y
- Dong, O. X., & Ronald, P. C. 2019. Genetic engineering for disease resistance in plants: Recent progress and future perspectives. Plant Physiology, 180(1), 26–38. 10.1104/pp.18.01224
- Lieber, M. R. 2010. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annual Review of Biochemistry, 79(1), 181–211. 10.1146/annurev.biochem.052308.093131
- Liang, F., Han, M., Romanienko, P. J., & Jasin, M. (1998). 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. https://doi.org/10.1073/pnas.95.9.5172
- Yang, W. (2010). Nucleases: Diversity of structure, function and mechanism. Quarterly Reviews of Biophysics, 44(1), 1–93. 10.1017/S0033583510000181
- Liu, Q., Yang, F., Zhang, J., Liu, H., Rahman, S., Islam, S., Ma, W., & She, M. (2021). Application of CRISPR/Cas9 in crop quality improvement. International Journal of Molecular Sciences, 22(8), 4206. 10.3390/ijms22084206