An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice

Abstract

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T₀ plants and 83.2% of T₁ plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T₁ progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.

Keywords: CRISPR/Cas9; homology-directed repair; marker-free; rice; stem-, shoot tip- and inflorescence-strong promoter (Pssi).

Figure 1

Identification of the stem-, shoot tip- and inflorescence-strong promoter Pssi. (a) The relative expression levels of OsSRABB compared with OsUFC1 in shoot apical meristem (SAM), inflorescences, seeds, leaves and seedling root are shown as normalized data (log2) from Rice eFP Browser. Bule indicates low transcript levels, and red indicates high transcript levels. (b) qRT-PCR analysis of OsSRABB in different tissues/organs of rice (Oryza sativa L.) variety Zhonghua11 (ZH11). The “V” and “R” following various tissues/organs represent vegetative and reproductive stages, respectively. OsUFC1 was used as the internal reference gene. Data are calculated from three biological replicates, and shown as mean ± SD, n = 3. (c) The distribution of cis-elements in different colors on the 858-bp promoter region of OsSRABB (Pssi).

Figure 2

Development of a marker-free system based onPssi-driving CRISPR/Cas9-mediated homology-directed repair (PssiCHMF tool). (a) Schematic diagram of the pYLPssi::Cas9 construct and the working model of the marker-free system. When CRISPR/Cas9-mediated homology-directed repair (HDR) occurs, the T-DNA carrying marker genes are excised, and the separated “GU” and “US” sequences are recombined into an intact GUS reporter gene. If excision is successful, the 1.1-kb recombined product is amplified by the GU-F and US-R primers, otherwise the band would be 1.3 kb, generated by GU-F and T35S-R. (b) PCR using primers (GU-F, T35S-R, and US-R) together to detect the excision of the marker gene cassettes in a heterozygous state (with one 1094-bp small band, generated by GU-F/US-R, and one 1277-bp large band, generated by GU-F/T35S-R) and non-excision (only with one 1277-bp large band, generated by GU-F/T35S-R) in pYLPssi::Cas9 T₀ plants. CK⁺, pCAMBIA1305 containing intact GUS; CK⁻, the pYLPssi::Cas9 construct. (c) A summary of the efficiencies of the marker gene excision by HDR in pYLPssi::Cas9 T₀ plants. The edited heterozygous marker-excision plants (11 plants) and non-excision plants (4 plants) are indicated.

Figure 3

PssiCHMF-edited recombined cells were accumulated during maturation in pYLPssi::Cas9 T₀ plants. (a) Quantification of SpCas9 and GUS transcript levels in various tissues and organs of pYLPssi::Cas9 T₀ plants. The “V” and “R” following different tissue and organ names represent vegetative and reproductive stages, respectively. OsUFC1 was used as the internal reference gene. Data are calculated from three biological replicates, and shown as the mean ± SD, n = 3. (b) Histochemical determination of GUS activity in different tissues and organs of pYLPssi::Cas9 T₀ plants at vegetative and/or reproductive stages. The calli were subcultured for 4 weeks. Bars = 0.5 cm.

Figure 4

The PssiCHMF system is a time-saving and efficient marker gene excision tool. (a) Heterozygous (with two bands, generated by GU-F/T35S-R and GU-F/US-R) and homozygous (only with one small band, generated by GU-F/US-R) marker gene excision plants were confirmed in pYLPssi::Cas9 T₁ lines by PCR to amplify the recombined products. Three independent transformed lines were used in this study. The homozygous excision plants are marked with red asterisks (*). CK⁺, pCAMBIA1305 containing intact GUS; CK⁻, the pYLPssi::Cas9 construct. (b) A summary of the efficiencies of the marker gene deletion by HDR in pYLPssi::Cas9 T₁ lines. The edited heterozygous (average of 26.7%) and homozygous (average of 55.6%) marker-excision plants are indicated.