CRISPR-Cas9-mediated gene editing is enabled by the same methods as the
production of genetically modified organisms. The only difference is that
the transferred DNA sequence is sorted out of the plant at the end of the
experiment, making the plant transgene-free. However, researcher Roman
Jerala of National Institute of Chemistry in Slovenia and colleagues say
that the insertion of the transgene at the earlier part of the experiment
can cause unwanted mutations. Also, further expression of the transgene in
the plant can cause off-target mutations. Therefore, the team proposed a
method to skip transgene production in gene editing.
The team transformed Cas9 enzymes and guide RNA sequence with
ribonucleoprotein into plant protoplasts using PEG-mediated transformation.
Results showed up to 25 percent mutation frequency in
FRI and PDS in the said species. They also observed positive correlation
between amount of CRISPR components and mutation rate. The team aims to
target other genes and study edited protoplast regeneration in the future.