Plant gene editing through de novo induction of meristems

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Abstract

Plant gene editing is usually carried out by delivering reagents such as Cas9 and sgRNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. Creating edited plants through tissue culture is often inefficient, requires considerable time, only works with limited species and genotypes and causes unintended changes to the genome and epigenome. We report methods to generate gene edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene editing reagents are delivered to somatic cells on whole plants. Meristems are induced that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene edited meristems sidesteps the need for tissue culture, promising to overcome a bottleneck in plant gene-editing.

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DNA replicons for plant genome engineering.

Nicholas Baltes, Javier Gil-Humanes, Tomas Cermak … (2014)

Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of plant genomes remains challenging due to ineffective methods for delivering reagents for genome engineering to plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activator-like effector nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA. In addition to the nuclease-mediated DNA double-strand breaks, gene targeting was promoted by replication of the repair template and pleiotropic activity of the geminivirus replication initiator proteins. We demonstrate the feasibility of using geminivirus replicons to generate plants with a desired DNA sequence modification. By adopting a general plant transformation method, plantlets with a desired DNA change were regenerated in <6 weeks. These results, in addition to the large host range of geminiviruses, advocate the use of replicons for plant genome engineering.

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Advancing Crop Transformation in the Era of Genome Editing.

Fredy Altpeter, Nathan Springer, Laura E Bartley … (2016)

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.

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A draft genome sequence of Nicotiana benthamiana to enhance molecular plant-microbe biology research.

Aureliano Bombarely, Hernan Rosli, Julia Vrebalov … (2012)

Nicotiana benthamiana is a widely used model plant species for the study of fundamental questions in molecular plant-microbe interactions and other areas of plant biology. This popularity derives from its well-characterized susceptibility to diverse pathogens and, especially, its amenability to virus-induced gene silencing and transient protein expression methods. Here, we report the generation of a 63-fold coverage draft genome sequence of N. benthamiana and its availability on the Sol Genomics Network for both BLAST searches and for downloading to local servers. The estimated genome size of N. benthamiana is 3 Gb (gigabases). The current assembly consists of approximately 141,000 scaffolds, spanning 2.6 Gb with 50% of the genome sequence contained within scaffolds >89 kilobases. Of the approximately 16,000 N. benthamiana unigenes available in GenBank, >90% are represented in the assembly. The usefulness of the sequence was demonstrated by the retrieval of N. benthamiana orthologs for 24 immunity-associated genes from other species including Ago2, Ago7, Bak1, Bik1, Crt1, Fls2, Pto, Prf, Rar1, and mitogen-activated protein kinases. The sequence will also be useful for comparative genomics in the Solanaceae family as shown here by the discovery of microsynteny between N. benthamiana and tomato in the region encompassing the Pto and Prf genes.