An efficient CRISPR/Cas9 system for simultaneous editing two target sites in Fortunella hindsii

Abstract

The CRISPR/Cas9 system is a revolutionary genome editing technique and has been widely used in numerous plants. For plants (e.g. citrus) with very low transformation efficiency, how to optimize gene editing efficiency and induce large-fragment deletion has been the focus of research. Here, we report that CRISPR/Cas9 induces efficient deletion of 16-673 bp fragments in the genome of Fortunella hindsii. The ability of two binary vectors, pK7WG2D and pMDC32, to introduce specific mutations into the genome of F. hindsii was evaluated. Double single guide RNAs (sgRNAs) were designed to achieve precise editing of two sites of a gene and deletion of fragments between the two sites. The construction of vectors based on Golden Gate assembly and Gateway recombination cloning is simple and efficient. pK7WG2D is more suitable for F. hindsii genome editing than the pMDC32 vector. Editing efficiency using the pK7WG2D vector reached 66.7%. Allele mutation frequency was 7.14-100%. Plants with 100% allele mutations accounted for 39.4% (13 100% allele mutation plants/33 mutants). The proportion of mutant plants with fragment deletion induced by this editing system was as high as 52.6% (10 fragment-deletion mutants/19 FhNZZ mutants). Altogether, these data suggest that our CRISPR/Cas9 platform is capable of targeted genome editing in citrus and has broad application in research on the citrus functional genome and citrus molecular breeding.

Figure 1

Process of assembly of Cas9 gene and two sgRNA expression cassettes. a and b Cloning of a single gRNA into pYPQ131A or pYPQ132A by the Golden Gate reaction and transformation in E. coli chemically competent cells. Esp3I is required for digesting pYPQ131A and PYPQ132A. c Assembly of two sgRNAs into the pYPQ142 acceptor; BsaI is required. d and e Assembly of the Cas9 gene and two sgRNAs into the T-DNA binary vector PK7WG2D (Gateway recombination); P35S initiates pcoCas9 gene expression, and kanR is the kanamycin resistance gene. gRNA1 and gRNA2 represent the guide sequence.

Figure 2

Targeted genomic editing of FhDUO1 by the CRISPR/Cas9 system. a Schematic illustration of the two guide sequences (sequences highlighted in red) targeting the FhDUO1 coding sequence. b Direct sequencing of PCR product chromatograms with overlapping traces demonstrating successful gene editing in the target regions of FhDUO1 in six representative T0 plants. pK7WG2D:FhDUO1 #3–8 represent six mutant plants for pK7WG2D:FhDUO1. The sequencing chromatograms from wild type (WT) served as the negative control. Red arrows indicate the positions where or from where the mutations occurred. c Detection of genomic mutations by T7 endonuclease I (T7E1) assay. The target fragments were amplified by PCR from genomic DNA extracted from transgenic plant leaves. #1–13 represent 13 mutant plants for pK7WG2D:FhDUO1. #(1) and #(2) represent two mutant plants for pMDC32:FhDUO1. Red arrows point to bands of expected size after T7EI digestion. +, PCR products were added; −, no PCR products were added. d Direct sequencing of PCR product chromatograms with overlapping traces demonstrating successful gene editing in the target region of FhDUO1 in two T0 plants. pMDC32:FhDUO1 #(1) and #(2) represent two mutant plants for pMDC32:FhDUO1. Red arrows indicate the positions where or from where the mutations occurred.

Figure 3

Targeted large-fragment deletions at FhNZZ in F. hindsii. a Schematic illustration of the two guide sequences (sequences highlighted in red) targeting the FhNZZ coding sequence. b PCR-based detection of deletions at FhNZZ. Red arrows indicate the deletion bands. The numbers indicate 51 transgenic plants for pK7WG2D:FhNZZ.  c Direct sequencing of PCR product chromatograms with overlapping traces demonstrated successful gene editing in the target regions of FhNZZ in three representative T0 plants. pK7WG2D:FhNZZ #2, #24, and #47 represent three mutant plants for pK7WG2D:FhNZZ. The sequencing chromatograms from wild type (WT) served as the negative control. Red arrows indicate the positions where or from where the mutations occurred. d Sequence confirmation of different lengths of deletions at FhNZZ induced by different sgRNA pairs. #2, #24, and #47 are mutant plants; deletions and insertions are highlighted in red and blue, respectively. Substituted nucleotides are highlighted in green. T-DNA binary vector is pK7WG2D.

Figure 4

Characterization of targeted editing in F. hindsii. a Mutation type analysis of the FhDUO1 gene. The frequency of the different editing events was calculated from the 137 mutation sites. b Mutation type analysis of the FhNZZ gene. The frequency of the different editing events was calculated from the 127 mutation sites. c Editing efficiencies of different allele types driven by the AtU6 promoter in F. hindsii. Figures in parentheses are the numbers of involved targets and sequenced sites. Nd, nucleotide deletion; Ni, nucleotide insertion; Ns, nucleotide substitution; Fi, fragment insertion; Fd, fragment deletion.

Figure 5

Partial phenotypes of FhDUO1 mutant plants induced by CRISPR/Cas9. a Leaf curling in duo1 mutant compared with wild type (WT). b The FhDUO1 mutation led to a longer flower peduncle and pedicel at the same developmental stage. c Pedicel longitudinal diameter statistics. Values represent mean ± standard deviation (WT, n = 100; FhDUO1 mutants, n = 100). d Schematic diagram of conserved motifs of downstream target genes of FhDUO1 elucidated by TBtools [27]. e Expression analysis of target genes downstream of FhDUO1 (mean ± standard deviation, n = 3). Asterisks indicate significant differences compared with WT (^**P < .01, Student’s t-test).