Asexual Gene Drive in Populus? Results from CRISPR/Cas9 Mutagenesis of Floral Genes for Genetic Containment

Gene flow from genetically engineered (GE) trees into feral or wild populations are significant obstacles to their use as a result of regulatory, public perception, and ecological concerns.  Loss-of-function mutations in a number of floral transcription factor genes can lead to sterility in diverse plant species, however such mutations are rare and generally recessive, thus are very difficult to induce via conventional tree breeding.  The recently rediscovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas system has proven to be a powerful directed-mutagenesis tool in many species, including trees.  The CRISPR-Cas induced mutations appear to be highly predictable and stable, and reversion should be extremely rare or impossible (e.g., when there are deletions of essential parts of coding regions).  We are testing the mutation efficiency of six nucleases targeting two essential floral genes in Populus.  The targets are the poplar orthologs LEAFY and AGAMOUS—well studied genes essential for both male- and female-fertility.  The nucleases have been stably transformed into hundreds of independent events that we are now analyzing for mutation rate and type.  Sequencing results from 300 events indicate that the rate of identical mutation in both LEAFY alleles is ~32% and in both AGAMOUS alleles is ~27%.  This high rate of identical biallelic mutations may be due to CRISPR-Cas-mediated gene drive (allelic conversion) during initial mutagenesis or mitotic growth.  Results from study of the full population will be presented.