High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns

Abstract

The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana, we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.

Keywords: CRISPR; Cas9; gene targeting; targeted mutagenesis.

Figure 1

Comparison of different Cas9 versions by the mutagenesis of TRY and CPC. (A) Structure of the Cas9 constructs and mutagenesis efficiency. The number of transformants obtained for each construct is shown (Transf. nb). For the constructs with intronized Cas9, which produced many plants with mutant phenotypes, transformants from a part of the trays were uprooted for phenotype evaluation (70–109 randomly selected transformants). pNos, nopaline synthase promoter; Bar, coding sequence of the Bar gene; Ocst, octopine synthase terminator; Rps5a, Arabidopsis ribosomal protein 5a promoter; hCas9, human codon-optimized Cas9 coding sequence; zCas9, Z. mays codon-optimized Cas9 coding sequence; zCas9i, the same sequence with 13 introns represented as six yellow boxes; zCas9io, a sequence variant of zCasi; F, FLAG tag; N, nuclear localization signal; Nost, nopaline synthase terminator; U6p, Arabidopsis U6 promoter; T/C, target sequence of the guide RNA for TRY and CPC genes; gRNA, the conserved region of the guide RNA; LB and RB, left and right T-DNA borders. (B) Picture of the primary transformants of two non-intronized Cas9 constructs, pAGM51523 and pAGM51535, and an intronized Cas9 construct pAGM51559. White arrows point to chimeric plants with leaves sectors with mutant phenotype.

Figure 2

Cas9 protein accumulation observed after transient expression in N. benthamiana leaves and stable transformation in Arabidopsis plants. (A) Accumulation of Cas9 upon the expression of different genes in N. benthamiana. Indicated Cas9 versions were expressed (under p35S control) in N. benthamiana by agroinfiltration. Tissues were used for protein extraction and immunodetection at 3 dpi. Ponceau staining of the membrane is shown as loading control. (B) Accumulation of Cas9 upon expression from different genes in stable Arabidopsis transformants. Proteins were extracted from the pools of leaf tissues from eight independent primary transformants expressing each Cas9 version (with sgRNAs targeting try and cpc; 5-week-old plants) and used for SDS–PAGE and immunodetection. Pools of the first four samples (Cas9 without introns) were prepared from six WT-like and two chimeric plants. Pools of transformants expressing intron-optimized Cas9 variants were prepared from plants with mutant phenotypes (try cpc; hairy). Ponceau staining of the membrane is shown as the loading control. (C) Detection of Cas9 in individual T₁Arabidopsis transformants. Proteins were extracted from the leaf tissues of individual T₁ transformants expressing either zCas9 or the intron-optimized zCas9i, both contained N- and C-terminal NLSs and were used for SDS–PAGE and immunodetection. Tissues exhibiting a WT-like phenotype were used for zCas9, and those with a try cpc-like phenotype were used for zCas9i. Ponceau staining of the membrane is shown as the loading control. (D) Subcellular localization of Cas9 versions carrying one or two NLSs. As in (A), except that fusions of Cas9 with mEGFP were expressed in N. benthamiana, tissues harvested 3 dpi were used for live cell imaging. The intron-optimized zCas9i gene was used for the expression of GFP-Cas9^NLS and ^NLSGFP-Cas9^NLS (NLS from SV40).

Figure 3

Comparison of different Cas9 versions in low-copy vectors by the mutagenesis of TRY and CPC. Structure of the Cas9 constructs and mutagenesis efficiency. The legend for the annotations is the same as in Figure 1. pNos, nopaline synthase promoter; Bar, Bar gene coding sequence; Ocst, octopine synthase terminator; Rps5a, Arabidopsis ribosomal protein 5a promoter; hCas9, human codon-optimized Cas9 coding sequence; zCas9, Z. mays codon-optimized Cas9 coding sequence; zCas9i, zCas9 sequence with 13 introns represented as six yellow boxes; zCas9io, sequence variant of zCasi; F, FLAG tag; Nost, nopaline synthase terminator; U6p, Arabidopsis U6 promoter; T/C, target sequence of the guide RNA for the TRY and CPC genes; gRNA, the conserved region of the guide RNA; LB and RB, left and right T-DNA borders.

Figure 4

Mutagenesis of floral homeotic genes using intronized Cas9 constructs. (A) Schematic representation of the constructs and cloning strategy. For each construct, two oligonucleotides were ligated into the cloning vector pAGM55261 digested with BsaI. The resulting constructs were transformed into Arabidopsis by the floral dip method. (B) Phenotypes of the wild-type control and transformants. (C) Estimation of the number of transformants with wild-type and mutant phenotypes.

Figure 5

Generation of large chromosomal deletions in Arabidopsis using the intron-optimized zCas9i. (A) General architecture of the constructs used for generating large deletions. sgRNAs (under the control of pU6-26) are transcribed in the opposite direction relative to zCas9i. FAST, pOle1:Ole1-tagRFP_tole1 (Shimada et al., 2010). (B) Schematic drawing of the RPP5 and RPP2 loci with sgRNA target sites (triangles; color code corresponds to [A]) and primer binding sites indicated. Genes are indicated by arrows with Arabidopsis gene identifiers, and a transposable element is shown as a rectangle. Not drawn to scale. (C) Screening of T₁ plants for candidate lines carrying a deletion at the RPP5 locus. T₁ plants were selected by resistance to BASTA and screened by PCR with the indicated primers for the occurrence of a deletion at the RPP5 locus. Thirty-four independent T₁ lines were screened and putative deletion lines are marked with arrowheads. A PCR product of ∼590–750 bp was expected for deletion lines. (D) PCR screening of segregants from T₂ families 14 and 15 shown in (C) for the isolation of Δrpp5 deletion line. Δrpp5 deletion alleles detected by PCR are shown at the top (oligonucleotides 1730/31; product size 592 bp; see also Supplemental Figure 11). The PCR product from a T₁ plant served as the control (ctrl). Wild-type RPP5 locus detected by PCR is shown at the bottom (1730/32; product size 606 bp). PCR products of Col DNA served as the control. (E) Summary of frequencies of large deletions in Arabidopsis generated by intron-optimized zCas9.

Figure 6

Frequency of small deletions in N. benthamiana generated by zCas9i. (A) Gene models of loci targeted for the induction of deletions by multiplex genome editing in N. benthamiana. Constructs used for editing the depicted loci had architectures similar to that shown in Figure 5A, except that zCas9i expression was driven by the 35S promoter. Roq1, GenBank entry MF773579.1 (Schultink et al., 2017); NRG1, Niben101Scf02118g00018, GenBank entry DQ054580.1; NPR1, NPR1-like gene, Niben101Scf14780g01001. (B) Genotyping of primary (T₀) transformants by PCR. For each transformation, results from 10 or 11 randomly chosen T₀ individuals are shown, and genotyping was conducted using oligonucleotides indicated in (A). Red arrows mark individuals lacking a PCR product corresponding to the wild-type fragment. Blue arrows mark individuals with multiple (>2) PCR products, which indicate somatic chimerism.

Figure 7

Site-targeted mutagenesis in C. roseus using the intronized Cas9. (A) Picture of a wild-type plant with flower, an in vitro-grown plant used for transformation, and a nodal segment with emerging hairy roots. plant. (B) Summary of the mutations generated by multiplexing in transgenic hairy root lines of CrJAM2 and CrJAM3. The genomic DNA of each line was extracted, the alleles were PCR-amplified and cloned into Escherichia coli (E. coli), and E. coli colonies were sequenced with the indicated primers. Most lines had two alleles with different mutations in both parental sequences (designated alleles A and B; C. roseus is a diploid species). In one line, a third allelic sequence was identified (allele C), probably showing two cell lineages with different mutations in one of the two homologs. In some cases, only one allele was detected (noted as homozygous). This may have been due to the repair of one of the two parental sequences by gene conversion using the other mutated homolog as the template.