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

Abstract

SUMMARYGenome 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 T 0 and Arabidopsis T 2 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.

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

“…The lower overall mutation frequencies compared with those observed in the NCR gene editing experiment are likely due to the significantly larger size of deletions being sought. An inverse relationship between Cas9-mediated deletion frequency and size has been observed previously in both mammalian and plant cells (Canver et al, 2014;Xie et al, 2015;Ordon et al, 2017).…”

“…A number of different toolkits for plant genome engineering have been published within the past two years (Xing et al, 2014;Xie et al, 2015;Ma et al, 2015;Lowder et al, 2015;Zhang et al, 2016;Liang et al, 2016;Vazquez-Vilar et al, 2016;Kim et al 2016;Ordon et al, 2017). However, all have limitations: Most do not offer flexibility in the gene editing platform (TALENs or CRISPR/Cas9); some reagents are optimized for a non-target organism (e.g., humans); typically, there is little choice in promoters for expressing targeting reagents; most systems are limited to a single vector type (e.g., T-DNA); applications are limited to NHEJ-mediated gene knockouts; and most reagents are validated in only one or two model species.…”

Non-host Resistance Induced by the Xanthomonas Effector XopQ Is Widespread within the Genus Nicotiana and Functionally Depends on EDS1

“…Petit Havana was used. Generation of the Nbeds1 mutant N. benthamiana line was described previously (Ordon et al, 2016). …”

“…The synthesized fragment did not contain internal Bsa I or Bpi I restriction sites, and codon usage was additionally altered to eliminate target sites of Cas9 nucleases used for generation of eds1 mutant plants (Ordon et al, 2016). The fragment was cloned into pAGM1287 yielding pJOG285, and subsequently assembled together with pICH51277, pICH50010, and pICH41432 in pICH47732 to yield pJOG296 (Engler et al, 2014).…”

Separate product from process: framing the debate that surrounds the potential uptake of new breeding technologies

“…Johannes Stuttmann (Martin Luther University of Halle-Wittenberg) concluded the meeting with a description of his work using GE to investigate the defence response in Arabidopsis. In addition, he described a set of dicot GE vectors developed by his group and made available to the community (Ordon et al 2016). These plasmids were generated using the golden gate cloning system and allowed for the facile multiplexing of four guide RNAs.…”