CRISPR/Cas9 or prime editing? – It depends on…

Genome editing (GE) emerges to become an indispensable tool in basic research and crop breeding. This owes especially to clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, the newest kind of site-specific nucleases derived from an ancient bacterial immune system against foreign DNA [1]. New improvements are published almost weekly, and basically, CRISPR/Cas-based GE technologies can be distinguished into two categories in terms of introducing either random or distinct mutations at the target site: the first category comprises CRISPR/Cas9 (but also Cas12 or other CRISPR type 1 systems) causing random mutations at the target site when double strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ). The second category consists of precise genome editing technologies such as prime editing [2], base editors [3] and CRISPR/Cas systems applied in combination with donor templates to repair DSBs by the technically more challenging homology directed repair (HDR) pathway [1]. Thus, researchers and breeders can soon rely on “educated guess” while deciding which change to introduce within their gene of interest (GOI). At this point, there are several choices: change can mean to correct a non-functional allele restoring a common phenotype or mimic natural variation detected in other species. Functional genomics comparing genomes and transcriptomes of different organisms allows the identification of suitable natural polymorphisms in genes relevant to plant–pathogen interactions. Successful applications are for example polymorphisms detected in the eIF4E gene from pea, which were mimicked in Arabidopsis to create virus resistance [4] or in promoters such as the rice bsr-d1 promoter where a single base change enhanced binding of MYBS1 to downregulate a peroxidase gene, thereby increasing H2O2 production and Blast resistance [5]. There is indeed a bright future for GE applications in the breeding stress resilient crops, especially in light of climate change which increases both abiotic and biotic stress situations. However, biotic stress might be easier to address because during co-evolution between host and pathogen very specific mechanisms were established rendering the host resistant or allowing the pathogen to overcome this resistance. The latter mechanisms deployed by pathogens largely involve so-called effectors, which can be proteins, RNAs or DNA having only one purpose which is to manipulate the host [6,7]. The high specificity underlying effector–target recognition allows deploying GE to prevent pathogen-caused resistance breaking. One example is the meanwhile wellestablished strategy to knock-out (KO) crop susceptibility factors, thereby increasing resistance [8]. These host factors are required by the pathogen to successfully colonize its host and upon their loss susceptibility will be reduced. However, GE is currently predominantly deployed in a way that causes random mutations in the target gene, and thus there can be deletions or insertions multiple of 3 bps, which cause no frame-shift mutation and thereby probably no visible change in the phenotype. With HDR or prime editing, it should be possible to induce specifically 1 or 2 bp insertions or deletions to guarantee a KO phenotype. This would be a clever way to prevent that several of the GE-induced mutations are without clear phenotype. But the KO of susceptibility factors is generally not free of side effects given that host genes do not just exist for manipulation by the pathogens, but have a certain physiological function during plant development. However, due to the degenerated nature of the genetic code it is also possible to induce tailored changes, which will not negatively affect crop physiology and cause a trade-off. One example is to mutate the promoter cis-elements targeted, e.g. by bacterial transcription activator-like effectors (TALEs) to induce the expression of susceptibility genes [9] or to rewrite target sequences of small non-coding RNAs (sRNAs) deployed by pathogens during  * Corresponding author: Dirk Schenke, Department of Molecular Phytopathology and Biotechnology, Christian-Albrechts-University Kiel, Hermann-Rodewald Straße 9, 24118 Kiel, Germany, tel: +49-431-880-3249, e-mail: d.schenke@phytomed.uni-kiel.de Open Life Sciences 2020; 15: 868–870