Targeted sequencing of poplar and eucalypt Cas9 transgenics for off-target mutations

CRISPR gene editing, while highly efficient in creating desired mutations, also has the potential to cause off-target mutations. This risk is especially high in clonally propagated plants, where editing reagents may remain in the genome for long periods of time or in perpetuity. We studied a diverse population of Populus and Eucalyptus trees that had CRISPR/Cas9-containing transgenes that targeted one or two types of floral development genes, homologs of LEAFY and AGAMOUS. One potential effect of leaving editing transgenes in the genome for long periods of time is a heightened potential for off-target mutations. Off-target mutations are those that occur due to CRISPR/Cas activity but are located at unintended loci. Due to the nature of gRNA binding and Cas complex formation, these are most likely to occur at sites similar to but divergent from (mismatched) the true target sites.To estimate the types and rates of rare off-target mutations, we used a targeted-sequencing approach that delivered high sequence depth, queried a very large number of potential off-target sites, studied plants where CRISPR/Cas had been present for more than two years, and examined a large number of insertion events.