AsianScientist (Nov. 2, 2015) – Scientists at the Institute for Basic Science’s (IBS) Center for Genome Engineering in South Korea have created a way to genetically modify plants using CRISPR-Cas9 without the addition of DNA. Their work has been published in Nature Biotechnology.
Because no DNA is used in this process, the resulting genome-edited plants could be exempt from current regulations governing genetically modified organisms (GMO) and given a warmer reception by the public. What makes this work different is that these genetic modifications look just like genetic variations resulting from the selective breeding that farmers have been doing for millennia.
“The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation,” explained IBS Director of the Center for Genome Engineering Kim Jin-Soo.
CRISPR is an acronym for clustered regularly interspaced short palindromic repeat, which refers to the unique repeated DNA sequences found in bacteria and archaea. The CRISPR-Cas9 gene editing system uses a single guide RNA (sgRNA) to identify and edit the target gene. 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.
To avoid the use of DNA, the researchers mixed purified Cas9 protein with sgRNAs targeting specific genes from three plant species to form preassembled ribonucleoproteins (RNPs). They then 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. They found that Cas9 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 percent. 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.
The researchers were also able to definitively show that Cas9 RNPs can be used to genetically modify plants, which Kim points out, “paves the way for the widespread use of RNA-guided genome editing in plant biotechnology and agriculture.”
The RGEN RNP process could enable the production of plants that are heartier and more suited to climate change in order to feed Earth’s increasing population. Currently European Union GMO regulations do not 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.
The article can be found at: Woo et al. (2015) DNA-Free Genome Editing in Plants with Preassembled CRISPR-Cas9 Ribonucleoproteins.
Source: Institute for Basic Science.
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