Source figure.
While exploring research internships last spring, I could have never guessed that I would get the chance to work on one of the most exciting advances in genomics since the sequencing of the human genome. This summer I was very fortunate to be selected to participate in a six-week research internship in Prof. Mueller’s laboratory, at the Boyce Thompson Institute for plant research, a research institute located on Cornell University’s campus. As an intern, I was involved in the field of bioinformatics, a field that combines biology and computer science to further research in biology. Working under the guidance of a fantastic post-doctoral researcher, Dr. Noe Fernandez, I focused on the so-called CRISPR/Cas-9 genome editing system.
The discovery of the CRISPR/Cas9 method in 2012 represented a revolutionary advance in genome editing [1]. Inspired by a bacteria’s remarkable ability to defend against viral DNA attacks, the CRISPR/Cas9 method results in a double strand break in a targeted DNA region. Once such a break occurs in a DNA region, two forms of natural repair can occur. In the presence of a suitable repair template for the gene region, Homologous Recombination (HR) repairs the DNA. Scientists can use HR for precise gene manipulations, such as inserting new segments of DNA. In the absence of a suitable repair template for the gene region, Non Homologous End Joining (NHEJ) repairs the DNA. NHEJ often results in either the insertion or deletion of nucleotides known as InDels. InDels are formed randomly, but frequently alter the Target Reading Frame of the gene and significantly alter the set of genetic instructions for amino acids, effectively silencing a gene. Because CRISPR/Cas9 can be designed to target a specific DNA region, the genome editing technique provides an exciting new method for studying the functionality of different genes.
Fig 1. Diagram of CRISPR/Cas9 system and functionality. Source [6].
The design of a CRISPR/Cas9 system requires two main functional groups: A Cas9 protein that cuts the targeted DNA strand and an sgRNA (single guide RNA) that guides the protein to specific locations in the genome. See Figure 1. A guide RNA sequence is comprised of a 20-nucleotide body conjoined with a specific a three nucleotide motif known as a Protospacer Adjacent Motif (PAM). By creating guide sequences specific to a certain location in a target gene, researchers can design powerful CRISPR systems to allow for gene modification as well as gene silencing.
Although the creation and implementation of CRISPR/Cas9 genome editing tools takes place in the laboratory, the design of these systems requires sophisticated techniques from bioinformatics, in particular to find optimal guide RNA sequences. One of the main difficulties that arises in the design of guide sequences is that, generally, such sequences also match with other, undesired locations in the genome, known as off-targets. Several algorithms have been developed to score guide RNAs effectiveness by a variety of experimentally determined factors to ensure minimal off-target matching, especially in other genes.
The focus of my summer research project was to extend the scope of currently published CRISPR tools, such as CCTop and CRISPR-P [2, 3]. With added functionality, researchers will have greater specificity when selecting sgRNAs. In particular, researchers will be able to analyze guide RNA positions within a gene, rewarding preference for RNAs near the 5’ end (the front end of a gene), and view whether they target specific gene domains for increased desirability. Additionally, researchers will have the option to design sequences intended to target multiple genes within a family. This task is made possible because of the ability of guide RNAs to match multiple locations and because related genes often share many conserved genomic regions. By extending guide RNAs to target several genes, scientists studying multiple related genes or genes from the same family will more easily be able to experiment with simultaneously silencing several genes.
After developing the software tools extending the existing methods, I was able to design multiple guide sequences for the Receptor Like Cytoplasmic Kinase (RLCK) gene family of the tomato plant; the gene family plays an important role in the plant’s immune responses. Figure 2 gives an example.
Figure 2. Optimal guide RNA coverage of RLCK VIII gene family.
My research will help Dr. Thomas Jacobs from Professor Martin’s Lab at BTI, a researcher studying the tomato protein kinase family by utilizing CRISPR/Cas9 structures. Tom plans to use CRISPR/Cas9 to create models for the tomato with different genes from the tomato kinase family silenced, so that other researchers can use these models to study the functionality of kinase genes and their impacts on plant pathogen interactions. His goal is to create a mutant collection for the protein kinases in tomato similar to the large mutant collections of Arabidopsis (a model legume that was the first plant genome sequenced) [4]. Overall, extending the functionality of current CRISPR tools allows for more specialized guide sequences to be obtained, enabling scientists a greater range of experiments when researching genes through genetic manipulation.
While the discovery of CRISPR/Cas9 represents an amazing breakthrough in genetics, there are some very real ethical concerns associated with the discovery. Researchers believe that the technique could be a great leap forward in preventing genetic disorders and deleterious genetic mutations; however, many people have begun to worry about whether altering genomes, especially in humans, is ethical and safe. A real concern is that with the ease of genetic manipulation, CRISPR could be used with nefarious intentions. In fact, some of these concerns have already become more pertinent, as scientists have started conducting research on human embryos using CRISPR. As this article describes [6], several Chinese research labs have started the study of the effect of CRISPR/Cas9 genome editing on basic cells of ‘non-viable’ human embryos. I believe that developments such as this one demonstrate the caution that that is needed when following promising discoveries. Although the discovery of CRISPS/Cas9 is clearly revolutionary in the world of science, future research needs to take into account the ethical concerns associated with genetic alteration. In particular, it will be important to ensure that genome editing will remain a driving force for good, for example, by helping us find new treatments for diseases or obtaining plants with better resistance to pathogens.
In the end, working as an intern at the Boyce Thompson Institute (BTI) proved to be a truly invaluable experience. Although I started the internship with a minimal background in bioinformatics and plant biology, through the amazing guidance of my mentor Dr. Noe Fernandez, I gained an immense amount of knowledge about these areas. Every day presented an array of different challenges, teaching me the rigor and dedication needed to conduct research. My experience over the six weeks showed me the excitement of being in the pursuit of discovery, and opened my eyes to the possibility of continuing to conduct research in the future. The opportunity to be imaginative, creative, and to think critically about a problem strongly appeals to my interest as an individual. I would like to extend my sincere gratitude to Tiffany Fleming, Nicole Waters Fisher, my mentor Dr. Noe Fernandez, Professor Lukas Mueller, and the entire Mueller lab for making my summer internship at BTI such an amazing and memorable experience.
Links
“Jennifer Doudna, a Pioneer Who Helped Simplify Genome Editing,” New York Times, May 11, 2015
Boyce Thompson Institute article by Patricia Waldron.
References
[1] Jinek, Martin, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, and Emmanuelle Charpentier. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337, 6096 (2012): 816-821.
[2] Stemmer M, Thumberger T, del Sol Keyer M, Wittbrodt J, Mateo JL (2015) CCTop: An Intuitive, Flexible and Reliable CRISPR/Cas9 Target Prediction Tool. PLoS ONE 10(4): e0124633. doi:10.1371/journal.pone.0124633
[3] Yang Lei, Li Lu, HaiYang Liu, Sen Li, Feng Xing, and Ling-Ling Chen. “CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants.” Molecular plant 7 (2014): 1494-1496.
[4] José M. Alonso, Anna N. Stepanova, Thomas J. Leisse, Christopher J. Kim, Huaming Chen, et al., and Joseph R. Ecker. Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana. Science 1 August 2003: 301 (5633), 653-657.
[5] Source figure: http://www.transomic.com/getattachment/0c3a5b99-05eb-43ee-a253-892eaf34b018/transEdit.aspx?maxsidesize=800
[6] Cyranoski, David, and Sara Reardon. Chinese scientists genetically modify human embryos. Nature (2015).
Stumbling into Science and Math 