The rice codon optimized Cas12a from Lachnospiraceae bacterium ND2006 containing two nuclear localization signals (NLS) at its N- and C- terminals were from previous report except with 3bp changes to remove 2 Bsp119I and one RsrII sites. This gene was driven by sugarcane Ubiquitin4 promoter (prSoUbi4) in two binary vectors. Tandem duplicate crRNA array (DR-S1-DR-S1) driven by OsU6 promoter was designed following Wang et al report. A 9 bp poly (T) short sequence was used to terminate the crRNA (OsU6-DR-S1) expression cassette. Comparing to base vector 24259, two extra expression cassettes are present in test vector 24277.
Plant Transformation and plant nursery
IR58025B rice was used for transformation. Agrobacterium-mediated transformation was performed according to the protocol reported previously. PMI positive plants were identified via selection medium containing mannose. Survived plants went through a TaqMan assay to check the T-DNA copy number and target sequence mutation of Xa13 promoter. Plants with single copy T-DNA insertion, backbone free and target site mutation of Xa13 promoter were sent to the greenhouse. Plants were grown in 170 × 150 mm pots filled with turf, peat moss and nutrition soil at a ratio 3:2:1 plus 40g Osmocote 3-4 months controlled-release fertilizer (17-7-12) per pot in greenhouse. Watering was managed via drip irrigation. Growth conditions were 30 ± 2°C days, 25 ± 2°C night, and the photoperiod was set to 12 hours day and 12 hours night.
TaqMan assay and sequence analysis of targeted mutations
TaqMan quantitative PCR (qPCR) assay was performed to determine the T-DNA copy number and target sequence mutation. Genomic DNA was extracted from leaf following the protocol of Promega Magbeads Plant genome extraction kit. Gene specific primers/probes were designed using the software PrimerExpress3.0 and synthesized from Life Technology. Real-time (RT) qPCR was performed in an ABI 7900HT real-time PCR system. Each 10 μl Real-time PCR reaction contained 5 μl 2x Sigma JumpStart Master Mix (Sigma-Aldrich Corporation), 3 μl DNA, 0.2 μl 50x Taqman assay stock each (final concentration: 300 nM for primers and 100 nM for probe) and 1.6 μl water. Real-time PCR conditions were as follows: 95 °C for 5 min; 40 cycles of 95 °C for 5 sec followed by 60 °C for 30 sec. The data were analyzed using the SDS 2.4 software.
The candidates screened by TaqMan assay were further confirmed by colony sequencing. The targeted regions were amplified with KOD-PLUS-Neo (Toyobo) and cloned into the pEASY vector (pEASY-Blunt Zero Cloning Kit, Transgen). Ten independent random clones were selected for Sanger sequencing (Life Technologies). The sequences were aligned to the wild-type in Vector NTI.
Seed color sorting
A fluorescence detector was used for seed color sorting. With the matching barrier filter glasses, the red fluorescent seeds could be easily sorted from the non-red fluorescent seeds. The number of red fluorescent and non-red fluorescent seeds in different E1 events was counted manually.
Xanthomonas oryzae pv. Oryzae (Xoo) strain PXO99 was cultivated on petri dish with YDC medium. Bacteria pathogen was scraped from petri dish to make suspension at 5 dpi with ddH2O and the suspension concentration was adjusted to OD 1.0 for inoculation. Rice plants were inoculated by leaf-clipping method at booting stage, and control planted were treated by ddH2O. Inoculated plants were incubated in growth chamber with 25℃ darkness condition for 24 hours, then change to normal rice plant growth condition. Lesion length and area were measured at 14 days post inoculation, and calculated mean and standard deviation to analyze Xoo resistance.
Gene expression analyses
Total RNA from leaf, anther, and root of rice was extracted with RNAprep pure Plant Kit (Tiangen Biotech, www.tiangen.com); 1 μg of total RNA was used for cDNA synthesis using the Superscript III first-strand system (Invitrogen) and the oligo-dT primer. Semi-quantitative RT-PCR was conducted as described by Zhou et al. PCR primers for Xa13 were 5’- ATGGCAGGAGGTTTCTTGTCC-3’ and 5’-AAGAAGCCGCCCACGTTC-3’; Primer sequences for OsAction (AY212324) control gene were 5’-GCAGAAGGATGCCTATGTTG-3’ and 5’- GGACCCTCCTATCCAGACAC-3’.