In this study, the authors explore using CRISPR-Cas9 in plants.

In this experiment, the

plant Arabidopsis was used. The authors targeted 14 loci using stackable arrays for gRNA

expression- this works by synthesizing groups of four bases and laying them on top of each

other. The authors used the GOLVEN gene family, which consists of 11 genes, for this

experiment. They cloned the stackable array so that it could become a binary vector and simulate

a wild-type gene in plants. Next, several plants of a single type were obtained, but editing rates

were not as high as expected in these plants when Cas9s function was confirmed. Since the cells

of a leaf are very diverse in function, the authors tested tissue from several plants in order to get

an accurate reading on the efficiency of Cas9. Cas9 was found to cut mostly at the -3 position,

and indels were taken into account instead of base substitutions. Cas9 detected mostly smaller

deletions and not larger deletions. Out of the 14 loci tested, only two did not have any deletions

detected, while the others had efficiencies ranging from 33% to 92%. Next, a computing program

was used to find any off-target sites, and 178 were evaluated. A hotspot for off-target indels was

found after evaluating 6 plants that had the same deletion in the same spot. In the discussion, the

authors conclude that SpCas9 is very efficient and accurate itself and can also be used to edit

plants as well. Other alternative gene-editing techniques are discussed, such as TALENs and

ZFNs, but in the end Cas9 still offers several advantages over these two methods. More research

has to be done to know how this will affect the GOLVEN family in other plant species.