Generation of chromosomal deletions in dicotyledonous plants employing a user‐friendly genome editing toolkit. (14th November 2016)

• Record Type: Journal Article
• Authors: Ordon, Jana; Gantner, Johannes; Kemna, Jan; Schwalgun, Lennart; Reschke, Maik; Streubel, Jana; Boch, Jens; Stuttmann, Johannes
• Is Part Of: Plant journal. Volume 89:Number 1(2017:Jan.)
• Page Start: 155
• Page End: 168
• Publication Date: 2016-11-14
• Subjects: CRISPR/Cas -- chromosomal deletion -- Nicotiana benthamiana -- Arabidopsis thaliana -- plant immunity -- EDS1 -- technical advance; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.13319
• Languages: English
• ISSNs: 0960-7412

Abstract

Significance Statement: In plants, genome editing has mostly been used to generate point mutations, but deletion alleles are often desirable. Here, we developed a toolkit for simple and efficient assembly of genome editing constructs and used it to generate deletions in both Nicotiana benthamiana and Arabidopsis. Small deletions (<100 bp) were recovered with relatively high frequencies in both species, while large deletions (up to 120 kb) were less frequent. Summary: 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.