CROPSR: A new tool to accelerate genetic discoveries
Written by Naphat Permpredanun '24
Edited by Jasmine Shum '24 and Jacqueline Cho '24
Figure 1 : CRISPR representation
According to the World Health Organization, “the number of people affected by hunger globally rose to as many as 828 million in 2021, an increase of about 46 million since 2020 and 150 million since the outbreak of the COVID-19 pandemic.” This report demonstrates the severity of food shortage and world hunger since it poses the threat of starvation to millions of people. While there have been solutions proposed such as policy changes regarding food distribution and infrastructure, the most prominent of these has been improving the current agricultural system and its technologies.The most notable contribution to this improvement is through genome editing using CRISPR/Cas9.
CRISPR/Cas9, or clustered regularly interspaced short palindromic repeats, is the well-known genome editing technique adapted from what bacteria use as an immune defense. According to CRISPR Therapeutic, the organization that uses gene editing to treat genetic diseases, researchers implement this technique by creating a small piece of RNA with a short "guide" sequence that attaches to a specific target sequence in a cell's DNA. This guide RNA also attaches to the Cas9 enzyme. When introduced into cells, the guide RNA recognizes the intended DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location, mirroring the process in bacteria. Through this process, DNA segments are edited to reveal new characteristics in the specimens such as drought resistant plants or mineral nourished plants. These creation would enhance the solution for world hunger, such as having a consistent food sources and decreasing malnourished people.
However, CRISPR/Cas9 gene-editing technology is more tailored towards meeting the needs of editing in mammalian genomes rather than complex crop genomes. As a result, several mistakes have been reported regarding inserting or deleting nucleotides. This problem may not be discovered until the plant matures which could be hard to detect especially among seasonal crops. To combat this gap between mammals and plants’ genetics, researchers from the Center for Advanced Bioenergy and Bioproducts Innovation invented a program called “CROPSR,” the first open-source software tool for genome-wide design and evaluation of guide RNA (gRNA) sequences for CRISPR experiments.
According to CROPSR’s developer Hans Müller Paul, "CROPSR provides the scientific community with new methods and a new workflow for performing CRISPR/Cas9 knockout experiments". This new workflow includes removing built-in biases in existing software tools that makes less mistakes in gene editing in plants . Because initially, these software tools are based on human or mouse genomes where multiple copies of genes are less common. Therefore, the existing tools often avoid gRNA sequences that hit the genome in more than one position to avoid causing mutations in places where they're not intended.
However, the goal for crops is often to mutate more than one position to knock out all copies of a gene, or mutating the DNA in a way that stops the gene's expression permanently. Previously, scientists had to design four or five mutation experiments to knock out each gene individually, requiring extra time and effort. With CROPSR’s framework, the developer creates a new machine learning model that would not avoid guides for repetitive genomic regions often found in plants, leading to a better handling of plants.
Moreover, CROPSR can improve the database of genomes from mammals to plants by generating a database of usable CRISPR guide RNAs for an entire crop genome. That process is computationally intensive and time-consuming, but researchers only have to do it once to build a database that can then be used for ongoing experiments as shown in Figure 2. This difference offers researchers an easier way to compare the target gene and the backbone compared to the traditional approach that requires a repetitive run of CRISPS through polygenes.
Figure 2 : Overview of a CRISPR experiment using CROPSR Timeline and steps of a typical CRISPR/Cas9 knockout experiment in a crop plant genome
CROPSR still contains some problems. For example, this program currently provides files that could be accessible by limited users based on the database system the users apply. CROPSR also has a problem with excessive computing power. This program must tackle the calculations on multiple genomes in polyploid plants, and some simplifications that are allowed in mammals, such as “knocking DNA” are not allowed in plants. Therefore, this program has to consider more factors, leading to high computing costs.
Both problems; however, are being addressed by the CROPSR team in the reports. For the database issue, the team will start implementing this program to allow more database systems to connect with the main database, which allows more users to access or add information about sequences of plants into CROPSR. For the computational power issue, the CROPSR team includes the development in the future as they mention that “future plans include a revision of the CROPSR code to provide better hardware scaling optimization”. This statement reflects that scaling optimization, or the code implementation that decreases the time used for computation through a computer system, will be used.
CROPSR is the new technology invented to solve the problem of genome editing in plants that contain more complex structures compared to mammals that CRISPR normally tackles on. This program increases the accuracy of detecting the guide DNA, or marker of the start of genome sequencing, which decreases a mistake in editing the genome. It also decreases the time finding the editing region compared to traditional CRISPR. With these benefits, this technology is deployed to support solving food shortage by creating more suitable plants in troubled crops.
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Figure 2 Reference: https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-022-04593-2/figures/4