Continual improvement of CRISPR-induced multiplex mutagenesis in Arabidopsis.

CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of the promoter driving Cas9 expression is critical to generate germline mutations. Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side by side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported. We genotyped more than 6000 Arabidopsis T2 plants to test seven promoters and six types of NLSs across 14 targets to systematically improve the generation of single and multiplex inheritable mutations. We found that the RPS5A promoter and bipartite NLS were individually the most efficient components. When combined, 99% of T2 plants contained at least one knockout (KO) mutation and 84% contained 4- to 7-plex KOs, the highest multiplexing KO rate in Arabidopsis to date. These optimizations will be useful to generate higher-order KOs in the germline of Arabidopsis and will likely be applicable to other CRISPR systems as well.

Accelerating wood domestication in forest trees through genome editing: Advances and prospects.

The high economic value of wood requires intensive breeding towards multipurpose biomass. However, long breeding cycles hamper the fast development of novel tree varieties that have improved biomass properties, are tolerant to biotic and abiotic stresses, and resilient to climate change. To speed up domestication, the integration of conventional breeding and new breeding techniques is needed. In this review, we discuss recent advances in genome editing and Cas-DNA-free genome engineering of forest trees, and briefly discuss how multiplex editing combined with multi-omics approaches can accelerate the genetic improvement of forest trees, with a focus on wood.