Dr. Raghuram Badmi:

I wish to convey my sincere thanks for this opportunity to share my views on the CRISPR-Cas9 or, more generally, genome editing technologies. Genome editing tools such as CRISPR-Cas9, clustered regularly interspaced short palindromic repeats - CRISPR associated protein 9, transcription activator-like effector nucleases, TALENS, and zinc finger nucleases, ZFNs, are all, in their basic forms, a pair of specific DNA scissors. They are specific for a particular DNA code, meaning that they can only recognize one specific stretch of a DNA sequence. If these scissors encounter a DNA sequence that does not match with their own DNA binding code, they cannot bind or cut that sequence.

These scissors scan the DNA, recognize and bind to their specific DNA sequence and perform the cut. Once there is a cut in the DNA, the cellular repair machinery recognizes the cut and initiates the repair process. Any cut in the DNA is a signal to the cell for recruiting the repairing tools at that site and completing the repair process. When the cell decides to repair the DNA, it mostly chooses a repair mechanism called non-homologous end joining, NHEJ. During the NHEJ repair process, a different DNA base - A, T, G or C - is added to the DNA 30% to 70% of the time, thereby changing the DNA code. The part of the DNA with the changed code loses its function and the plants that have these different bases are selected. This is how the editing of the genome or genome editing is achieved. This type of editing comes under the site-directed nuclease type-1, SDN-1, technology where it does not require an outside DNA and occurs as a result of an error-prone DNA repair mechanism of the cell. This method would be the first-choice for any researcher because the only aim here is to break the DNA at the desired location, then the cell takes over for the rest of the process. One example of SDN-1 is soybean oil with no trans-fat, with high oleic oil and low linolenic oil content developed by Calyxt using TALENS. This is the first commercially-available gene-edited plant product in 2019. The second example is camelina, the plant in the mustard family used for oil with enhanced omega-3 oil. It was developed by using CRISPR by Yield10 Bioscience and cleared by the United States Department of Agriculture, USDA, in 2017.

However, sometimes only breaking the DNA is not enough. When there is a need to improve the function of a certain gene, a stretch of DNA which is functional, it might require adding, deleting or changing specific DNA bases at a particular DNA location. To do such precise editing, a repair DNA template is introduced, along with the scissors - DNA scissors plus repair DNA template. The repair DNA template has the correct bases that we want to add or delete and the DNA address for where it should go and stick to. When we do this, the cell chooses a different repair mechanism called homology directed repair, HDR, about 5% to 10% of the time at best. During HDR, the cell copies the DNA bases present in the repair DNA. The copied DNA segment replaces the original DNA segment and completes the repair process.

A modified version of CRISPR-Cas9 known as base editing is emerging. Base editing, as the name suggests, is used to convert the specific base in the DNA from A to G or from C to T, without the need for a repair DNA template. These types of editing come under the category of site-directed nuclease type-2, SDN-2, technology that includes making small but precise edits to the DNA in the order of a few bases, which in most cases are sufficient to improve functions of the gene. Examples of base editing are high nitrogen use efficiency in rice by changing one base cytosine, C, to thymine, T, using cytidine base editor, and high yield in rice by changing adenine, A, to guanine, G, using adenine base editor and simultaneously C to T at specific places of SPL genes.

Site-directed nuclease type-3 technology involves inserting entire genes or DNA fragments, which can comprise more than 1,000 base pairs. SDN-3 is used to provide novel functionalities to the plants that are not present before. An example of SDN-3 is golden rice, a variety of rice engineered to produce beta-carotene, pro-vitamin A, to help combat vitamin A deficiency.

SDN-1 and SDN-2 both mimic natural mutations caused by chemicals or irradiation. However, unlike other mutations that occur randomly in the entire genome of an organism, SDN-1 and SDN-2 are very precise, which is almost everything we could ask for.

On the next sheet of my written opening statement I have some additional information for all the members to go through.