New Toolkit for Plant Genome Engineering

The Science

Develop plant genome engineering techniques to allow efficient gene knockouts, replacements, altered transcriptional regulation, and multiplexed modifications.

  • A suite of direct and modular cloning vectors was developed to build TALEN-or CRISPR/Cas9-based editing targeting multiple genes and containing different promoters, reporters, selectable markers, and codon-optimized genes.
  • Vector construction is facilitated by an online tool that allows choice of different vector modules for different functions, either in monocot or dicot plants.

The Impact

  • The toolkit was tested for deletion mutagenesis targeting single or multiple genes, taking advantage of polycistronic transcripts with multiple guide RNAs (gRNA) in a single construct.
  • Up to 6 genes were successfully knocked out using multiplexed vectors, and a 58 Kbp genomic region containing 5 genes involved in nitrogen fixation was deleted in a single event.
  • The new toolkit will accelerate genome engineering of bioenergy crops, providing a flexible and comprehensive set of gene-editing options.

Summary

Researchers report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Their reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible.

References

Cermák, T., S. J. Curtin, J. G.-Humanes, R. Čegan, T. J.Y. Kono, E. Konečná, J. J. Belnato, C. G. Starker, J. W. Mathre, R. L. Greenstein, and D. F. Voytas. 2017. “Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants,” The Plant Cell 29(6), 1196–1217. DOI:10.1105/tpc.16.00922.