Precise and heritable gene targeting in rice using a sequential transformation strategy

Abstract

Gene targeting (GT) is a powerful tool for modifying endogenous genomic sequences of interest, such as sequence replacement and gene knockin. Although the efficiency of GT is extremely low in higher plants, engineered sequence-specific nucleases (SSNs)-mediated double-strand breaks (DSBs) can improve GT frequency. We recently reported a CRISPR-Cas9-mediated approach for heritable GT in Arabidopsis, called the "sequential transformation" strategy. For efficient establishment of GT via the sequential transformation method, strong Cas9 activity and robust DSBs are required in the plant cells being infected with Agrobacterium carrying sgRNA and donor DNA. Accordingly, we generated two independent parental lines with maize Ubiquitin 1 promoter-driven Cas9 and established sequential transformation-mediated GT in the Japonica rice cultivar Oryza sativa Nipponbare. We achieved precise GFP knockin into the endogenous OsFTL1 and OsROS1a loci. We believe that our GT technology could be widely utilized in rice research and breeding applications.

Keywords: CRISPR-Cas9; DNA methylation; OsFTL1; OsROS1a; epigenetics; gene targeting; genome editing; genome engineering; rice.

Graphical abstract

Figure 1

Generation of rice parental lines (A) Outline for the sequential transformation method in rice. GT donor constructs with sgRNAs targeting genes of interest and containing a selection marker were transformed into the parental line, which is already transgenic for an incidental intergenic region-targeting sgRNA and for Cas9 driven by the ZmUbq1 promoter. T0 transgenic lines were selected with Basta, and individual T0 plants were analyzed. The positive lines were used for further experiments. (B) Rice parental line ZmUbq1 pro:Cas9 construct. Schematic represents the transfer DNA construct for the rice parental line, which harbors Cas9 driven by the ZmUbq1 promoter, an sgRNA driven by the OsU6 promoter, and the hygromycin selection marker HptII gene driven by the CaMV 35S promoter. (C) Mutation efficiency and Cas9 expression in transgenic T0 rice. The mutation ratio was determined by qChop-PCR (gray bar). The relative Cas9 expression value was calculated from OsAct1 (black bar). The error bars indicate standard deviations (n = 3). (D) Relationship between Cas9 expression and mutation among ZmUbq1 transgenic T0 lines. The Cas9 expression level and mutation ratio in the 26 independent ZmUbq1 pro:Cas9 transgenic T0 lines were plotted. The mutual fund is shown by the coefficient determination (R²). (E) Mutation of the target locus genotype in selected T0 transgenic rice plants. (F) Mutation of the target locus and Cas9 genotype in T1 transgenic rice plants. The mutation genotype was determined by RFLP. The PCR products were digested with SpeI. Asterisks indicate Cas9 transgene-free homozygous or biallelic mutant rice plants. NP, nontransgenic Nipponbare as a control; di, SpeI digested; un, SpeI undigested controls.

Figure 2

GFP knockin into the OsFTL1 locus (A) Schematic of the OsFTL1-GFP donor construct and endogenous target locus. The donor construct contains an in-frame GFP sequence with endogenous OsFTL1 harboring 800 bp homology arms (Figure S1A). Horizontal line represents the probe for Southern blotting (Figure S1A). (B and D) PCR and Southern blot hybridization genotyping of OsFTL1-GFP T0 (B) and T1 (D). (C) Sequence confirmation of the precise GFP knockin for T0 OsFTL1-GFP. Silent mutations are indicated by green rectangles.

Figure 3

GFP knockin into the OsROS1a locus (A) Schematic of the OsROS1a-GFP donor construct and endogenous target locus. The donor construct contains an in-frame GFP sequence with endogenous OsROS1a harboring 1,000 bp homology arms (Figure S1B). Horizontal line represents the probe for Southern blotting (Figure S1B). (B and C) PCR genotyping and Southern blot hybridization of OsROS1a-GFP T0 (B) and T1 (C). (D) qRT-PCR for OsROS1a expression in the T1 OsROS1a-GFP plants. The error bars indicate standard deviations (n = 4). (E) Evans blue staining of transversally sectioned mature grains. The scale bar indicates 0.5 mm.

Figure 4

Epigenetic effects at the GT target OsFTL1 locus Individual locus bisulfite sequencing analysis. (A) Bisulfite sequencing at the 5′ and 3′ homology arms is shown for OsFTL1-GFP T0 rice plants. Purple, total C methylation; red, CG methylation; blue, CHG methylation; green, CHH methylation. (B) Individual bisulfite sequencing at the knockin GFP sequence. At least 20 independent clones were sequenced for each sample for bisulfite sequencing analysis. Sequencing results were analyzed using Kismeth.