Genome-edited plants, without DNA

What makes this work so groundbreaking is that these genetic modifications look just like genetic variations resulting from the selective breeding that farmers have been doing for millennia. IBS Director of the Center for Genome Engineering Jin-Soo Kim explains that "the targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation."

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeat, which refers to the unique repeated DNA sequences found in bacteria and archaea. CRISPR is now used widely for genome editing. What's crucial in genetic engineering is for the gene editing tool to be accurate and precise, which is where CRISPR-Cas9 excels. CRISPR-Cas9 uses a single guide RNA (sgRNA) to identify and edit the target gene and Cas9 (a protein) then cleaves the gene, resulting in site-specific DNA double-strand breaks (DSBs). When the cell repairs the DSB, the resulting fix is the intended genetic edit.

The beauty of this research is that the IBS research team has elevated the process and no longer uses DNA, being unshackled from GMO regulations. To do this, purified Cas9 protein was mixed with sgRNAs targeting specific genes from three plant species to form preassembled ribonucleoproteins (RNPs). The IBS team used these Cas9RNPs to transfect several different plants including tobacco, lettuce and rice to achieve targeted mutagenesis in protoplasts. To test the efficacy of this process, the team delivered Cas9 RNPs to the protoplasts of the test plant species, and foundCas9 RNP-induced mutations 24 hours after transfection.These newly cloned lettuce cells showed no mosaicism which led the researchers to believe that the RGEN RNP may have cleaved the target site immediately after transfection and the indels occurred before cell division was completed.

Finally, the team demonstrated that RGEN-induced mutations were maintained after regeneration. Using a Cas9 RNP, they disrupted a gene in lettuce called Brassinosteroid Insensitive 2 (BIN2) which regulates the signaling of brassinosteroid, a class of steroid hormones responsible for a wide range of physiological processes in the plant life cycle, including growth. They found that after cell division the lettuce cells maintained the disruption of the gene with a frequency of 46%. Importantly, there were no off-target indels. They grew full plants from the seeds of these genome edited and regenerated plants,which had the mutation from the previous generation. They were able to definitively show that Cas9 RNPs can be used to genetically modify plants, which Jin-Soo Kim points out,"paves the way for the widespread use of RNA-guided genome editing in plant biotechnology and agriculture."

The IBS team's technique of genome editing without inserting DNA could be revolutionary for the future of the seed industry.The RGEN RNP process will enable us to produce plants that are heartier and more suited to climate change in order to feed Earth's increasing population. Currently European Union GMO regulations don't allow for food with added DNA. Since the Cas9 RNP technique does not use DNA, it may be able to avoid being in violation of these rules. In addition, using Cas9 RNP is cheaper, faster and more accurate to apply to plants than previous breeding techniques (like radiation-induced mutations). Large agribusiness companies have been able to afford the time and money necessary to create seeds for genetically modified food, but the Cas9 RNP technique could allow for a more decentralized gene-edited seed production industry.

This process is ready for use to bolster plant output and create heartier crops in foods like tomatoes and lettuce. The application of the Cas9 RNP gene editing technique could be the next step in ending food shortages.