CRISPR/dCas-mediated gene activation toolkit development and its application for parthenogenesis induction in maize

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems can be engineered as programmable transcription factors to either activate (CRISPRa) or inhibit transcription. Apomixis is extremely valuable for the seed industry in breeding clonal seeds with pure genetic backgrounds. We report here a CRISPR/dCas9-based toolkit equipped with dCas9-VP64 and MS2-p65-HSF1 effectors that may specifically target genes with high activation capability. We explored the application of in vivo CRISPRa targeting of maize BABY BOOM2 (ZmBBM2), acting as a fertilization checkpoint, as a means to engineer parthenogenesis. We detected ZmBBM2 transcripts only in egg cells but not in other maternal gametic cells. Activation of ZmBBM2 in egg cells in vivo caused maternal cell-autonomous parthenogenesis to produce haploid seeds. Our work provides a highly specific gene-activation CRISPRa technology for target cells and verifies its application for parthenogenesis induction in maize.

Keywords: CRISPRa; ZmBBM2; apomixis engineering; egg cell; maternal haploid.

Figure 1

Evaluating transcriptional activation of CRISPRa (a1–a6) constructs in maize mesophyll protoplasts. (A) Core components of the CRISPRa vectors. dCas9, Streptococcus pyogenes Cas9 lacking endonuclease activity (mutations D10A, H840A); ERF2m, modified transcriptional activator from Arabidopsis ETHYLENE RESPONSE FACTOR2; HSF, human heat-shock factor 1; MS2, bacteriophage coat proteins; NLS, nuclear localization signal; P65, NF-κB trans-activating subunit P65; pU6, ZmPolIII U6-2 promoter; pUBI, maize Ubiquitin1 promoter; sgRNA1.0, routine sgRNA of Cas9; sgRNA2.0, sgRNA scaffold containing two MS2 stem loops; T, terminator; T2A, insect virus Thosea asigna self-cleaving peptide (18 amino acids); VP64, four copies of the herpes simplex virus early transcriptional activator VP16. (B) Schematic diagram of the three components of the a4–a6 systems with MS2 connected to the transcriptional activation domains P65 and HSF1. (C and D) Activated ZmDPS1 expression, as determined by qRT–PCR. (C) Schematic diagram of the ZmDPS1 locus. The first transcription start site was designated as +1. (D) Fold activation of ZmDPS1 expression when the sgRNA targets the sequence between –1 and –100 from the transcription start site using a1–a6. The maize Ubiquitin gene (NCBI GenBank: U29159.1) was used as the reference. Relative expression levels were calculated using the 2^−ΔCt (threshold cycle) method. (E) Fold activation of ZmDPS1 expression as a function of sgRNA location with the a5 system. (F–H) Validation of the a5 system with the genes (F)ZmTrxh, (G)ZmES2, and (H)ZmRCP1, which have low transcript abundance in maize mesophyll protoplasts. All values are means ± SEM with n = 3 biological replicates. An unpaired t-test was used to determine significant differences (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Figure 2

Isolation of single female gamete cells and verification of the CRISPRa5 system in egg cell (EC) protoplasts. (A–H) Micromanipulation and isolation of maize female gamete cells at the single-cell level. (A) Schematic diagram of maize ovary structure. AC, antipodal cell; CC, central cell; EC, egg cell; ES, embryo sac; SC, synergid cell. (B) Isolated maize ovary showing the exposed ES (red arrow). (C) Manual micromanipulation to separate the ES from the ovary under a stereomicroscope. (D) Obtaining single cells from the ES via partial enzymatic digestion. (E–H) Isolated ACs (E), SCs (F), CCs (G), and ECs (H). (I and J) Targeted activation of a stably transformed aleurone-specific DsRED cassette expressed in the EC in vitro. The DFP line was previously constructed and harbors a DsRED expression cassette driven by the barley aleurone-specific LTP2 promoter (Dong et al., 2018). (I) Schematic diagram of the CRISPRa5 vector targeting LTP2pro:DeRed2 with an effector cassette driven by the EC-specific promoter of Arabidopsis DD45 (Steffen et al., 2007). (J) Ectopic expression of DsRED in an isolated EC, as revealed by DsRED fluorescence. The vector (I) plasmid was delivered into the EC via electroporation, and fluorescence was observed 24 h after transient transformation. The DsRED signals were detected under a confocal microscope. Bright, brightfield. Bars: 50 μm.

Figure 3

Targeted activation of ZmBBM2 in egg cells in vivo. (A) Constructed vector of CRISPRa5^BBM (a5^BBM). 3×FLAG, 3 tandem FLAG epitope tags; Bar, BlpR gene. Other components are the same as those shown for CRISPRa5 in Figure 1A. (B) ddPCR identification of transformed copy numbers. The nine independent transformants were EA13, EA14, EA15, EA19, EA21, EA22, EA28, EA34, and EA36 in the T₃ generation. The a5^BBM amplitudes were scored with the FAM probe, and ZmADH1 reference amplitudes were scored with the HEX probe. Top left panel, amplitudes of the targets and the reference; top right panel, calculated a5^BBM copy numbers; bottom panel, typical droplets with the a5^BBM, ZmADH1, and a5^BBM/ZmADH1 amplitudes and blank amplitudes (left lower quadrant) of line EA19 plotted in four quadrants separated by two vertical crossing red lines. The corresponding droplet numbers are shown in each quadrant. (C–F) Quantification of gene expression in non-pollinated female gametophytes. (C and D) mRNA expression level of a5^BBM(C) and ZmBBM2(D) were scored in isolated ovaries. Quantifications were converted into expressed copy numbers per 1 μg total RNA. Each sample used 20 ovaries for RNA extraction. (E) ddPCR identification of mRNA expression among isolated ECs, ACs, SCs, and CCs from line EA19. (F)In situ hybridization identification of a5^BBM and BBM2 expression in egg cells in vivo. Feulgen staining was used to visualize ovule development. Immunohistochemical assays with alkaline phosphatase-labeled anti-FLAG antibody or BBM2 RNA probe were used for a5^BBM and ZmBBM2 detection, respectively. (G and H)ZmBBM2 and a5^BBM mRNA expression time course from 0 to 9 days after pollination in the embryo sac. Each sample used 20 embryo sacs for RNA extraction.

Figure 4

Apomixis phenotypes caused by the targeted activation of BBM2 in vivo. (A) Double embryo sacs in EA19 ovaries. Bars: 50 μm. (B–E) Efficiency of maternal haploid generation in engineered lines. (B) Screening of maternal haploid kernels produced from the cross EA19×ZC01^DFP. (C) Representative flowering plants and mature ears. (D) Cell DNA contents in haploid plants (right) and control diploid plants (left) determined by flow cytometry. (E) Efficiency of haploid generation across nine CRISPRa5^BBM lines.