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

First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.

“…Due to the construct integration within the genome, products developed by gene editing and other new genetic technologies must be subjected to GMO regulations in many countries [ 26 , 39 ]. For this reason, researchers are trying to develop new strategies to circumvent DNA integration, such as with the CRISPR/Cas9 delivery into protoplasts using RNP [ 28 ], a complex consisting of the recombinant Cas9 nuclease and the CRISPR RNA (crRNA) transcribed in vitro.…”

“…CRISPR RNP-based genome editing offers the opportunity to produce edited plants by means of DNA-free approaches, opening new perspectives for breeding purposes and potentially better acceptance by consumers as compared to classic GMOs (genetically modified organisms) [ 26 ]. In spite of these advantages, this technology has scarcely been applied so far to woody species: from the literature, we can only mention the reports on apple [ 15 , 27 ], grapevine [ 27 ] and pine [ 28 ].…”

Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation

“…Promoter sequences of snoRNA U6 and U3 genes (from here on abbreviated as pU6 and pU3, respectively) were identified in the genomes of a broad range of plant species, followed by the testing of their applicability for genome editing in the corresponding species. Four pU6 sequences were identified in the cucumber genome [ 167 ]; three pU6s were identified in cotton [ 168 ]; two pU3s and two pU6s were identified in grapevine ( Vitis vinifera ) [ 169 ]; six pU6s and one pU3 were identified in maize [ 170 ]; one pU6 was identified in stiff brome ( Brachypodium distachyon ) [ 171 ]; one pU6 was identified in Douglas fir ( Pseudotsuga menziesii ), which was used for CRISPR/Cas9 editing in radiate pine ( Pinus radiata ) [ 172 ]; one pU6 was identified in liverwort ( Marchantia polymorpha ) [ 173 ]; and one pU6 was identified in spreading earth moss ( Physcomitrium patens ) [ 174 ], in addition to others. Despite the wide variety of pU6/pU3 sequences described, changing from a widely used pU6/pU3 (e.g., pAtU6, pMtU6, pGmU6, or pOsU3) to a species-specific pU6/pU3 version does not guarantee improved genome editing efficiency, similar to what has been observed regarding the uncertain effects of Cas codon optimization.…”

From Genome Sequencing to CRISPR-Based Genome Editing for Climate-Resilient Forest Trees

“…The proof of concept for the CRISPR/Cas9 application has been established in several fruit tree species such as citrus [112,113], apple [114,115], grape [116], coffee [117], kiwifruit [118], cacao [119], pomegranate [120], walnut [121], and pear [115]. However, CRISPR-mediated genome editing in forest trees has been mainly achieved in poplar [122], and, for the last three years, in the tropical tree Parasponia andersonii [123], Eucalypts [124], rubber tree [125,126], Monterey pine [127], and European chestnut [128]. For evaluating CRISPR in new tree study systems, several types of the engineered SpCas9 gene sequences with nuclear localization signals and designed with codon optimization for human (hSpCas9, Addgene #42230 [11]), for Arabidopsis thaliana (aSpCas9, Addgene #61433, [129]), for rice (oSpCas9, Addgene #53064 [130]), for grasses including higher GC content at the 5 terminal region (gSpCas9, Addgene #106331 [131]), or even the original coding sequence from Streptococcus pyogenes have been successfully used.…”

“…Alternative transformation methods can also be considered to deliver RNPs while bypassing the regeneration steps, including de novo meristem induction [208]. Besides, genome editing that is mediated by direct delivery (i.e., by particle bombardment) of Cas9 RNP has recently been applied to edit the gene for glucuronic acid substitution of xylan 1 (GUX1) in Pinus radiata, the most extensively planted exotic conifer species [127]. Using the RNP approach, somatic embryogenic cells were successfully mutated at the target site (with 22-33% efficiency), however, producing only monoallelic plantlets.…”