Genome-Edited Plants
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 germ line-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.
