Peasy blunt zero vector

Double-stranded RNA (dsRNA) synthesis was conducted based on the cloned NlSP7 sequences. A nucleotide sequence (around 300 bp long) specific to the target gene was cloned into the pEASY® Blunt Zero vector (TransGen Biotech, Guangzhou, China) and submitted for sequencing to IGE Biotechnology Ltd. (Beijing, China). Aequorea victoria green fluorescent protein (GFP) was used as a control. PCR products were used as templates for dsRNA synthesis using a MEGAscript T7 transcription kit (Thermo Fisher, Waltham, MA, USA). Supplemental Table S1 shows the specific primers used to generate these DNA templates.
Newly molted 1-day-old brachypterous female adults were first anesthetized with carbon dioxide for 15 s and then injected with approximately 100 ng of dsRNA using a Nanoject II Auto-Nanoliter Injector (Drummond Scientific, Broomall, PA, USA). After injection, the treated BPHs were reared on RH rice at 27 ± 0.5 °C at a relative humidity of 70 ± 5% for 3 days. The injected BPHs were used in the experiments.

We performed bisulfite sequencing PCR (BSP) to measure the DNA methylation level. We extracted the genomic DNA from the fruit and injected it with ddH₂O and 5′-Aza using FastPure Plant DNA Isolation Mini Kit (Vazyme, Nanjing, China). We used a DNA Methylation Kit (CW2140M, CoWin Biosciences) to convert bisulfite. BSP was performed using Phanta® Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China). The BS primers are listed in Additional file 1: Table S4. All fragments generated by BSP were introduced into the pEASY-Blunt Zero vector (CB501-01, TransGen Biotech) using ClonExpress® II One Step Cloning Kit (Vazyme, Nanjing, China). For each sample, ten individual clones were sequenced. The CyMATE software (http://www.cymate.org/) was used to analyze the sequencing results and measure DNA methylation levels.

mRNA transcription was performed on a linearized DNA template containing T7 promoter and rabbitfish HNF4α cDNA sequence (GenBank: JF502073.1) using mMESSAGE mMACHINE^® T7 Ultra Kit (Ambion, Austin, TX) according to the manufacturer to generate capped mRNA with a poly(A) tail. Plasmid pEASY-Blunt-Zero-HNF4α was constructed with pEASY-Blunt-Zero vector (TRANS Gen, China) and HNF4α cDNA, which was used as a template for a linearized DNA template above with T7 promoter primer and antisense primer of HNF4α containing termination codon in a pfu-PCR reaction (Table 1). PCR procedure were one cycle (94°C for 3 min), 30 cycles (94°C for 30 sec, 65°C for 30 sec, 72°C for 1 min 30 sec), one cycle (72°C for 10 min). Finally, the product containing HNF4α mRNA was purified with MEGAclear^TM Kit (Ambion, Austin, TX), and stored in -80°C for further transfection into rabbitfish primary hepatocytes, described previously [28 (link)]

The in-frame deletion of dndE in pJTU1238 was constructed by two-step PCR. In the first step, the upstream and downstream fragments of dndE were generated by PCR using the primer pairs ΔdndE-1/ΔdndE-2 and ΔdndE-3/ΔdndE-4, respectively (Table 1). ΔdndE-2 and ΔdndE-3 are chimeric primers. A mixture of two purified PCR products, which overlapped by 40 bp, served as the template for a ligation PCR using primers ΔdndE-1 and ΔdndE-4. An 1,877-bp product containing the dndE deletion fragment was generated and cloned into the pEASY-Blunt Zero vector (TransGen Biotech), yielding plasmid pWHU675. This plasmid was confirmed by sequencing analysis. The NsiI-XhoI fragment of pWHU675 containing an in-frame deletion of dndE (corresponding to amino acid nos. 14 to 98; red in Figure 1) was inserted into the corresponding sites in pJTU1238 to generate the plasmid pWHU676.

The expression vector was constructed in two steps. The first step involved creation of the AtU6-sgRNA vector by ligation of the double stranded sgRNA with a linearized intermediate expression vector (cut by BbsI, New England BioLabs, Ipswich, MA, USA) containing the promoter of Arabidopsis thaliana U6 (AtU6). The intermediate expression vector was modified from the pEASY-Blunt-Zero vector (TransGen, Beijing, China) by adding AtU6 followed by restriction recognition sites of BbsI and XbaI (Figure 1B). The double stranded sgRNA used in ligation was prepared by slowly annealing together the following two complementary oligoes: sgRNA-F, 5′-GATTGTTTGTTGTGTCCGGGACCA-3′ and sgRNA-R, 5′-AAACTGGTCCCGGACACAACAAAC-3′ (the underlined nucleotides are the adaptor sequences complementary to the sticky ends of the intermediate expression vector after BbsI digestion) using the following temperature steps in a PCR machine: 95 °C 2 min, 70 °C 10 min, 55 °C 10 min, 40 °C 10 min, and 25 °C 10 min.
The second step was to move the AtU6-sgRNA fragment from the AtU6-sgRNA vector to the CRISPR/Cas9 vector. To do that, both vectors were digested completely with KpnI and XbaI. The purified AtU6-sgRNA fragment was then ligated with the linearized CRISPR/Cas9 vector to construct the final 35S-Cas9-AtU6-sgRNA gene editing vector (Figure 1C).

P. oxalicum 114-2 and P. oxalicum RE-10 were stored in our laboratroy. E. coli DH5α and pEASY-Blunt Zero vector from TransGen (Beijing, China) were used for propagation of plasmids and gene clone. The plasmid pPICZαA and host P. pastoris X-33 from Invitrogen (Carlsbad, CA, USA) were employed for the β-xylosidase gene expression. All strains and plasmids used in this study shown in supplementary Table S3.

Genomic DNA was extracted from etiolated rice seedlings by using a DNeasy plant mini kit (QIAGEN). Genomic RUPO, including a 2.8-kb promoter, the coding sequence, and 0.8-kb 3’UTR, was PCR-amplified with KOD-Plus-Neo DNA polymerase (TOYOBO) and cloned into the pEasy-blunt-zero vector (TransGen). To visualize the tissue expression pattern of RUPO::GUS, the 2.8-kb native RUPO promoter was fused to the pPLV15 vector via LIC cloning [54 (link)]. For genetic complementation, Genomic RUPO was fused into a modified pCambia1300 vector. The recombinant constructs were confirmed by DNA sequencing, then used for transforming rice callus induced from mature embryos.