Genome editing with CRISPR/Cas9 in Pinus radiata (D. Don)

Abstract Background To meet increasing demand for forest-based products and protect natural forests from further deforestation requires increased productivity planted forests. Genetic improvement of conifers by traditional breeding is time consuming due to the long juvenile phase genome complexity. modification (GM) offers opportunity make transformational changes in shorter frames but challenged current genetically modified organism (GMO) regulations. Genome editing, which can be used generate site-specific mutations, rapidly implement targeted improvements globally regulated a less restrictive way than GM technologies. Results We have demonstrated CRISPR/Cas9 editing P. radiata targeting single-copy cell wall gene GUX1 somatic embryogenic tissue produced plantlets edited tissue. generated biallelic INDELs with an efficiency 15 % using single gRNA. 12 transgenic was when two gRNAs were deletions up 1.3 kb identified. However, regenerated plants did not contain large had nucleotide insertions at one target sites. assessed use ribonucleoproteins (RNPs) their ability accomplish DNA-free P . radiata. chose hybrid approach, RNPs co-delivered plasmid-based selectable marker. A two-gRNA strategy 33 %, INDELs, including deletions. Using RNP found also present plantlets. But, all monoallelic. Conclusions generation monoallelic coniferous tree system plasmid expressed Cas9 gRNA respectively. This opens apply modify key traits interest.