Historically, traditional breeding methods took advantage of genetic variability to enhance crops through a long process of crossing and selecting. This timely and strenuous process pushed scientists to find better technology to shorten and improve the process. One such way, and most promising, is the technology of genome editing using CRISPR/Cas9

The CRISPR/Cas (clustered regularly interspaced short palindromic repeat) system evolved as an adaptive immune response in bacteria and archaea to defend against invading viruses. The bacteria capture snippets of invading viral DNA and create DNA plasmids known as CRISPR arrays, which are then used for memory of the virus. If the virus attacks again, the bacteria rapidly produce RNA from the CRISPR arrays to target the DNA and recruits Cas9 enzyme to create double stranded breaks (DSBs) in the DNA, disabling the virus.

In plant breeding, this technology can be exploited for developing precise edits within a plant’s genome to improve the crop. Using the same understanding, DNA targeting specific gene loci of interest and the Cas9 enzyme can be introduced into various organisms to create DSBs in the region of interest to a breeder.

The DSBs can be repaired by two separate pathways or the combination of both. The non-homologous end joining pathway (NHEJ) is error-prone and frequently introduces random small deletions or insertions at the junction of newly rejoined DNA. The homology-directed repair pathway (HDR) is precise and stimulated by the availability of the homologous DNA repair template (DRT) that surrounds the double stranded break. Both pathways, are exploited for different interests, NHEJ for generating the loss-of-function of a gene or knock-out mutants, while HDR can assist in allele/gene replacement or more precise modifications such as tagging genes of interest.

The application of this technology is increasingly viewed as vital due to rising threat of global climate change and the rise in diverse agricultural, environmental and ecological stresses. So far, many different CRISPR/Cas9 kits were developed to target mutagenesis at specific genome loci, transcriptome regulations, epigenome editing, base editing and precise gene replacement and tagging in plants and the use of these kits continues to increase.

At NRGene, our goal is to develop the ability to replace any DNA fragment with the desired version allowing breeders to efficiently introduce elite alleles into commercial cultivars. This is difficult to achieve through common crop breeding practices, and requires time-consuming and labor-intensive activities. Using CRISPR/Cas9 mediated HDR pathway of DSBs has the potential to mitigate those difficulties and introduce desired traits within only 2-3 generations in the absence of any undesirable traits – something that cannot be achieved using conventional methods. Thus, the precise replacement of an existing allele with an elite allele is the holy grail of crop breeding and genetic improvement.