EMPLOYING AN RNA-MODIFYING ENZYME TO EDIT BOTH RNA AND DNA FOR AGRICULTURAL APPLICATIONS

In the coming decades a growing global population, combined with the changing climate and other environmental stresses, will place a significant burden on the global demand for low-cost food and energy. This necessitates more efficient and innovative methods to produce the food and energy required to feed and fuel the planet. It is becoming more evident that traditional methods of improving and enriching crops and livestock will be insufficient to provide low-cost food for the world market especially for third-world countries. A new strategy must be developed to produce a higher yield of crops and livestock in the changing climate and stresses. One strategy is to introduce new traits in crops and livestock to increase yields and production, which might include attributes like faster growth rate and/or the ability to tolerate environmental stresses such as drought, disease, pests, and climate change. Traditionally, plant and animal breeders have used conventional methods in selective breeding programs to genetically improve plant and livestock species. While these conventional breeding techniques have improved crop plants and livestock, the rate of implementation is slow and will not meet the coming global demand for more food and energy. Therefore, a more efficient and precise method to edit the genetic material of organisms would be indispensable in order to insert new genes, to correct disease-causing genes, to activate or inactivate genes (knock-in or knock-out) in order to provide the desired traits. Today, one of the most common ways to genetically modify many diverse organisms is to employ the use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. This CRISPR/Cas9 system utilizes a bacterial nuclease (Cas9) together with a single guide RNA (sgRNA) that base-pairs with a region of DNA targeted for cutting (by the Cas9 nuclease). The Cas9 cleaves both strands of the DNA generating a double-stranded break. The system can be used to cut out a specific gene generating a knock-out, where the two strands of DNA in the genome are joined together by non-homologous end joining (NHEJ). Or if a new gene is presented with matching flanking sequences, the cell can utilize it DNA repair machinery to splice in a new gene by homology-directed repair (HDR) creating a knock-in trait. While this CRISPR/Cas9 system is a powerful method to create new genetic traits, it still has some limitations and shortcomings: e.g., it can cut DNA only near a requisite PAM sequence, also the technique can create unwanted insertions or deletions (indels) in the genome, and Cas9 is not efficient in changing or mutating a single nucleotide polymorph (SNP), the most abundant cause of genetic disorders. Many agriculturally important traits are associated with SNP variations. Altering SNPs is also an important means to improve agronomic characteristics of crops. Therefore, generation of point mutations at specific sites associated with important agronomic traits is of great value in molecular breeding. One of the goals of this proposal is the develop a new tool that the geneticist can employ to change a single nucleotide at a precise location in the genome without cutting the DNA, which causes unwanted indels. This proposed system will repurpose an RNA editing enzyme called Adenosine Deaminase acting on RNA (ADAR), to base-edit specific adenosines changing them to inosine, a base that behaves like guanosine in base-pairing interactions. Our lab recently determined the three-dimensional crystal structure of ADAR bound to dsRNA. This structure revealed the base-flipping mechanism, where the targeted adenosine is flipped out of the RNA double helix into the active site of the ADAR enzyme. The structure led us to hypothesize that ADARs edit bases in regions of double-stranded RNA and not dsDNA because the binding of ADAR is specific to A-form double helix, as observed in dsRNA. Since dsDNA is a B-form double helix, it will not bind to ADAR. However, an DNA/RNA hybrid does base-pair in an A-form double helical structure and preliminary data show that ADARs can indeed deaminate adenosines of the DNA strand of a DNA/RNA hybrid. This suggests that ADARs could be commissioned to reverse the most common disease-causing SNP, (G-to-A) in DNA, to revert back to the wildtype G base without cutting the DNA. Many recent publications have shown the importance and necessity of this type of editing in crop plants. A second goal of this project is to exploit the normal targeting activity of ADAR enzyme, in editing RNA. Because many people are concerned with GMO foods in their diet, if some genetic traits can be modified by changing the RNA, which translates into modified gene products, without actually editing DNA, the product of this technique would not designated as "Genetically Modified" because the DNA is not altered. The aim in this goal is to administer modified RNAs (resistant to nucleases), to base-pair with selected targets of mRNA to form regions of dsRNA that can be base-edited with ADARs. This would create modified RNAs with modified properties without permanent genetic changes. This aim would have more applications in animal livestock because RNAs would have to be administered to the organism by injections and rely on the native ADAR enzyme found in all animals.