Targeted gene insertion is the most challenging type of genome editing in plants. Its success depends on multiple factors, including transformation and regeneration processes, efficiency of genome editing components delivery, copy number of the repair template and its availability at the DSB site, size of the DNA sequence to be inserted, presence or absence of a selectable marker gene in the repair template, and HDR efficiency at a given target site. In addition, gene insertion experiments usually have high attrition rate of generated events due to the complexity of the insertion, plant chimerism, transmission to the next generation, and ability to segregate intended edits from helper genes (e.g. Cas9, gRNA, and selectable marker). Different approaches have been suggested and tested to overcome some of these issues and increase the overall success rate of targeted gene insertions: particle bombardment and Agrobacterium transformation, delivery of editing components as DNA, RNA, and ribonucleoprotein (RNP), suppression of NHEJ and activation of HDR pathways, viral replication to increase repair template copy number, linear and circular repair template molecules, tethering repair template to Cas9-gRNA RNP complexes, inducible, cell cycle-specific and developmental promoters, intragenomic and in planta approaches, and exploiting different insertion mechanisms – HDR, NHEJ, homology mediated end joining (HMEJ), and microhomology mediated end joining (MMEJ)54,55,56. However, despite new genome editing approaches and better understanding of underlying mechanisms of DSB repair, the progress in developing a simple, robust, and reliable process for targeted gene insertion in plants at a higher efficiency has remained limited.
Agrobacterium-mediated transformation is the preferred method for delivery of editing components into the plant cell57. This system is reproducible, amenable for a broad variety of crop species (both monocots and dicots) and cost effective since it doesn’t require special equipment and associated consumables. In addition, Agrobacterium-mediated delivery is less invasive, has lower attrition rate during regeneration stage58,59, and results in lower frequency of potential chromosomal rearrangements observed in particle bombardment experiments60,61. The major limitation of the Agrobacterium-mediated approach is a low number of T-DNA molecules transmitted into the plant cell during infection resulting in low copy number of repair template and, therefore, low frequency of targeted gene insertion. This disadvantage can be overcome by repair template copy number increase using geminivirus replication system56. However, this approach requires a more complicated vector design, results in uncontrolled DNA replication, may lead to unintended random integration of DNA into the genome, and can result in lower regeneration frequency35.
Due to low frequencies of genome editing via HDR, targeted insertion experiments require hundreds of regenerated events to recover plants with the desired outcome. Many important crop species, especially their elite genotypes, have low regenerability and are extremely challenging for complex HDR-based edits. A rapid maize transformation system48 that relies on expression of two morphogenic genes, Bbm and Wus262, allows recovery of transgenic plants from transformed immature embryos at high frequencies. The use of morphogenic genes has also significantly improved transformation efficiency of elite maize genotypes62, allowing a faster trait product development. Furthermore, it has been suggested that DSB repair via homologous recombination occurs predominantly during the late S, G2 and early M phases of the cell cycle, while being actively suppressed at G1 stage63,64,65,66. Consequently, efficient HDR-based genome editing is largely restricted to actively proliferating cells. Therefore, an additional benefit of using morphogenic genes in genome editing experiments is associated with an effect of BBM and WUS2 proteins on stimulation of cell division, providing an HDR-friendly cellular environment.
Another improvement of maize Agrobacterium-mediated transformation has been accomplished by developing a new ternary vector system that utilizes an optimized accessory plasmid pVIR953. This system increased transient T-DNA delivery and recovery of stable callus events, resulting in six-to-seven-fold improvement over conventional random transformation using the plasmid pSB1 in an elite maize inbreds. The combination of morphogenic genes and new ternary vector design resulted in high transformation efficiency, increased number of T-DNA molecules transmitted, and high frequency of plant regeneration. Implementation of this enhanced Agrobacterium transformation protocol allowed us to generate HDR-based insertion events at target site TS45 with frequencies up to approximately 3% (Table 1). However, further optimization of Agrobacterium-mediated gene insertion was advantageous.
Target sites flanking repair template was described in intragenomic gene targeting experiments36,37,38, where they were used to release the repair template from random integration loci. This design has an additional advantage as linear repair template potentially increases frequency of HDR-based gene insertion42,43. It might be even more advantageous considering recent results indicating that damaged DNA might be transported to specific loci at the nuclear for further repair67. Target sites flanking repair template have been used in recent particle bombardment experiments in Arabidopsis and rice25,45,46,47. However, a positive effect of this vector design on frequency of gene insertion could not be verified as no controls were provided in these experiments. Contrary, introduction of flanking target sites has resulted in 2-5-fold increase of integration frequencies relative to circular plasmids in human culture cells44. In our experiments, direct comparison of T-DNA vectors with and without target sites flanking repair templates, consistently demonstrated an approximately 3-fold increase of targeted gene insertion frequency, resulting in about 8–10% frequency of HDR-mediated gene insertion events based on the number of T0 plants analyzed (Table 1).
Target site TS45 used in this study was identified as one of the best sites with high HDR frequency in previous particle bombardment experiments15. Selection of this site was relevant for this study as it allowed to generate high number of HDR-based gene insertion events, compare results of different experiments and the corresponding controls, and draw reliable conclusions.
Another important factor that can significantly affect the outcome of gene insertion experiments, but rarely discussed, is the event attrition rate. For example, in this report, the frequency of HR1/HR2 junction qPCR positive events (approximately 8–10%) drops by approximately 50% after long PCR analysis. Most likely, this is the outcome of the concurrent action of HDR and NHEJ pathways often resulting in co-integration of various DNA molecules (vector DNA and/or genomic DNA sequences) into the target site19,68. In addition, our results indicate that attrition rate may depend on different parameters, including T-DNA vector configuration, such as position of selectable marker gene, regenerants survival rate, plant fertility, and chimerism. In this report, the combined attrition rate ranges from 64 to 80%, with the highest lost observed in the experiment with selectable marker gene outside the repair template (Table 2).
In summary, the advancements in Agrobacterium-mediated maize transformation48,53,62 combined with optimized vector design, enabled efficient targeted gene insertions in maize. Our experiments showed reliable and reproducible results for two genotypes with different transformability, two different genes, and for constructs with a selectable marker both inside and outside repair templates. These results open new opportunities for accelerated precision breeding in a wide range of crop species amenable to Agrobacterium-mediated transformation.