Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly genome editing toolkit

Genome editing facilitated by Cas9-based RNA-guided nucleases (RGNs) is becoming an increasingly important and popular technique for reverse genetics in both model and non-model species. So far, RGNs were mainly applied for the induction of point mutations, and one major challenge consists in the detection of genome-edited individuals from a mutagenized population. Also, point mutations are not appropriate for functional dissection of non-coding DNA. Here, the multiplexing capacity of a newly developed genome editing toolkit was exploited for the induction of inheritable chromosomal deletions at six different loci in Nicotiana benthamiana and Arabidopsis. In both species, the preferential formation of small deletions was observed, suggesting reduced efficiency with increasing deletion size. Importantly, small deletions (<100 bp) were detected at high frequencies in N. benthamiana T₀ and Arabidopsis T₂ populations. Thus, targeting of small deletions by paired nucleases represents a simple approach for the generation of mutant alleles segregating as size polymorphisms in subsequent generations. Phenotypically selected deletions of up to 120 kb occurred at low frequencies in Arabidopsis, suggesting larger population sizes for the discovery of valuable alleles from addressing gene clusters or non-coding DNA for deletion by programmable nucleases.