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

Abstract

Background: Our 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.

Results: We 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.

Conclusions: The SM series of CRISPR/Cas9 vectors enables the rapid generation of transgene-free, genome edited plants for a diversity of functional studies.

Keywords: Arabidopsis thaliana; CRISPR/Cas9; Genome editing; NLR genes; Natural variation.

Fig. 1

SM (super module) Destination binary vectors and sgRNA shuffle-in vectors. a Schematic representation of SM Destination binary vectors. Plant codon optimized Cas9 (pcoCas9) is driven by the promoter of UBQ10 (proUBQ10). Transcriptional termination sequences from rbcS. Blue-white selection strategy with LacZ cassette. Seed coat expressed red fluorescence from At2S3:mcherry cassette as transgenic plant selection marker. Not drawn to scale. b Schematic representation of sgRNA shuffle-in vectors. sgRNA including 20-bp target sequences and shared sgRNA sequences transcribed by A. thaliana U6 promoter. c Overhang sequences used for Golden gate cloning in generating the final binary vectors. The numbers listed in left column are indicated in a and b

Fig. 2

Workflow of using SM vectors

Fig. 3

An example of inherited deletions in different accessions. a Schematic representation of targeted gene At3g04220 (U17), with locations of sgRNA target sites and primer binding sites for genotyping. b Deletions of At3g04220 in Col-0 reference accession, as detected by PCR with primers designated in a. Nineteen individual T1 plants were tested. Expected size of PCR product for deletion is 404 bp. c Alignment of sequence of PCR product for deletion in b (red) with wild-type sequence, which is only partially shown. d Deletions of At3g04220 in T1 plants from natural accessions, as detected by PCR; number of pooled plants in square brackets. 1: Col-0 #1 sample in b; 2: TueSB30-3 [9]; 3: Nie1-2 [7]; 4: WalHaesB4 [13]; 5: Rue3.1-31 [6]; 6: TueWa1-2, TueV-13, and HKT2.4 [5] (transformation efficiency is low for these 3 accessions). e Inheritance of At3g04220 deletion in 28 transgene-free Col-0 T2 plants, progeny of #1 T1 plant in b. Several plants show only the mutant band (top), indicating that they are homozygous for the deletion

Fig. 4

Frequencies of deletions and inheritance in T2 generation. a Deletion frequencies in T1 plants for the 34 out of 53 targeted genes where deletions were detected (Additional file 2: Table S1). b Frequencies of deletion inheritance in T2 for the 17 genes whose deletions were inherited (Additional file 2: Table S1)