An efficient CRISPR vector toolbox for engineering large deletions in Arabidopsis thaliana

Abstract

BackgroundOur knowledge of natural genetic variation is increasing at an extremely rapid pace, affording an opportunity to come to a much richer understanding of how effects of specific genes are dependent on the genetic background. To achieve a systematic understanding of such GxG interactions, it is desirable to develop genome editing tools that can be rapidly deployed across many different genetic varieties.ResultsWe present an efficient CRISPR/Cas9 toolbox of super module (SM) vectors. These vectors are based on a previously described fluorescence protein marker expressed in seeds allowing identification of transgene-free mutants. We have used this vector series to delete genomic regions ranging from 1.7 to 13 kb in different natural accessions of the wild plant Arabidopsis thaliana. Based on results from 53 pairs of sgRNAs targeting individual nucleotide binding site leucine-rich repeat (NLR) genes, we provide a comprehensive overview of obtaining heritable deletions.ConclusionsThe SM series of CRISPR/Cas9 vectors enables the rapid generation of transgene-free, genome edited plants for a diversity of functional studies.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0330-7) contains supplementary material, which is available to authorized users.

Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system

“…By inducing two DSBs within a single chromosome, it is possible to obtain deletions or inversions. Although a number of papers on chromosomal deletion formation have been recently published (Ordon et al ., ; Durr et al ., ; Wu et al ., ), there has not been a study on efficient heritable inversion formation for plants till now. We demonstrated in this study that inversions up to 18 kb can be induced using the Cas9 ortholog from Staphylococcus aureus at different locations in the Arabidopsis genome with ease.…”

“…In plants, on the other hand, most reports focused on the induction of deletions and their inheritance into the next generation. Deletions were induced in Arabidopsis thaliana using different promoters for stage‐ or tissue‐specific expression of Cas9, obtaining a deletion size of up to 13 kb with high frequencies of heritable events or up to 120 kb with low frequencies (Ordon et al ., ; Durr et al ., ; Wu et al ., ). For stage‐specific expression, the egg cell promoter is commonly used for early DSB induction to create heritable mutations, large deletions and gene‐targeting events in Arabidopsis (Wang et al ., ; Durr et al ., ; Wolter et al ., ).…”

From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas

“…In comparison to mammals, applications in plants were mainly focused on the induction of deletions and the transmission of these events into the next generation. Small deletions up to 1 kb can be achieved quite easily in Arabidopsis and other plant species, but interestingly, it was also possible to induce deletions up to 120 kb with low frequencies in Arabidopsis, and stage specific expression of the Cas9 protein promotes the possibility of deletions to enter the germline (Ordon et al 2017;Durr et al 2018;Wu et al 2018). Furthermore, somatic deletion formation with a size of up to 245 kb using CRISPR/Cas was effectively achieved in other plant species like rice, tobacco and Medicago, although the inheritance was only shown for deletions in Medicago and rice (Zhou et al 2014;Gao et al 2015;Č ermák et al 2017).…”

Oligomerization of NLR immune receptor RPP7 triggered by atypical resistance protein RPW8/HR as ligand

“…To generate genome editing vectors, plasmids with an A. thaliana codon optimized Cas9 were assembled using the GreenGate system (Wu et al, 2018a); Cas9-free transgenic seeds were selected by mCherry fluorescence in their seed coats.…”