A single donor cassette enables site-specific knock-in at either the αAmy3 or αAmy8 locus in rice cells via CRISPR/Cas9

Abstract

CRISPR/Cas9 gene editing is widely used to manipulate gene expression and integrate transgenes into specific target sites, making it a powerful tool for recombinant protein expression. In this study, we generated a single donor cassette for CRISPR/Cas9-mediated knock-in at either the αAmy3 or αAmy8 locus in rice cells. The transgene was inserted downstream of the promoter and first exon of the rice αAmy3 or αAmy8 genes, which are highly expressed under sugar-starved conditions in rice suspension cultures. We constructed a simple vector with the homologous intron sequences of the αAmy3 and αAmy8, along with rice codon-optimized recombinant receptor binding domain (rcRBD) of the SARS-CoV-2 spike protein, a functional domain responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells. Using this construct, rcRBD was successfully integrated into the intron 1 of either the αAmy3 or αAmy8 genes. As a result, rcRBD expression was driven by endogenous αAmy3 or αAmy8 promoter-signal peptide. Under the control of αAmy3-signal peptide, rcRBD proteins was detected in both the soluble cellular protein fraction and culture medium, whereas expression driven by the αAmy8 promoter-signal peptide was exclusively detected in the culture medium of rice suspension cells. The highest secreted protein yield of rcRBD in the rice culture medium under the control of αAmy8 endogenous promoter reached 20.7 mg/L, demonstrating a production efficiency comparable to that driven by the endogenous αAmy3 promoter.

Keywords: αAmy3 promoter; αAmy8 promoter; CRISPR/Cas9; Intron; Rice suspension cells; Single donor cassette.

Fig. 1

Schematic diagram of the recombinant receptor-binding domain (rcRBD) of the SARS-CoV-2 spike protein, inserted into αAmy8 and/or αAmy3 intron 1 using the CRISPR/Cas9 system. Two CRISPR plasmids were constructed, each comprising a Cas9 expression cassette (Cas9) driven by the Ubiquitin promoter (Bui) and a hygromycin resistance gene (Hat) under the control of the Nos promoter (NosP). One plasmid carried the αAmy8 E8 sgRNA (sg), while the other contained the αAmy3 E3 sgRNA. The donor plasmid includes rcRBD flanked by E8 and C3 sgRNA target sites, which consists of the intron 3′ splicing site (3′ ss), rcRBD coding region, and Nos terminator (NosT). Upon co-bombardment of the CRISPR and donor plasmids, the 3′ss-rcRBD-NosT cassette is integrated into either the αAmy8 or αAmy3 intron 1, resulting in expression units driven by the respective endogenous αAmy8 or αAmy3 promoters and signal peptides. LB, left border; RB, right border; 35ST, 35S CaMV terminator; UbiIn, Ubiquitin intron (in plasmid); E1, Exon 1; Ex2, Exon 2; E3, Exon 3 (in genomic DNA)

Fig. 2

Generation of rcRBD knock-in rice cells. A PCR-based genotyping of rcRBD knock-in rice callus cell lines. Specific primer sets were used to amplify Cas9, rcRBD, αAmy3-rcRBD, αAmy8-rcRBD, and ACT1 as a reference gene. B DNA sequencing analysis of two αAmy3-rcRBD and four αAmy8-rcRBD CRISPR-mediated knock-in rice cell lines. PCR-amplified DNA fragments were subjected to Sanger sequencing to examine their 5′-junction sequences. Dark grey characters represent rice genomic DNA sequences upstream of the C3 sites, while light grey for upstream and downstream of the E8 site; blue and orange characters indicate E8 and C3 site sequences, respectively, with underlines indicating protospacer adjacent motif (PAM) sites. Red characters represent the artificial 3′ splice site. Green characters indicate the AscI restriction enzyme adaptor, and light green “ATG” represents the start codon of the rcRBD sequence. Purple characters represent NosT sequences; gold characters indicate the inserted sequences. Negative (−) and positive (+) signs denote nucleotides deleted and inserted at the intron target sites, respectively. Dots (…) indicate sequence continuation, and the double arrowhead (< + >) in gold represents a 361-nt insertion. Wild-type (WT) sequences were used as references for alignment with the knock-in rice cell line sequences, with introns shown in lowercase and exons in uppercase

Fig. 3

Characterizations of rcRBD knock-in rice cell lines. A Genotype analysis of rcRBD knock-in suspension cell lines. Suspension cell lines were cultured from two rcRBD knock-in cell lines, including rcRBD insertion into αAmy8 intron 1 (rcRBD-32) and αAmy3 intron 1 (rcRBD-42). PCR-based genotyping was performed using specific primers for rcRBD, αAmy3_E1-rcRBD, αAmy8_E1-rcRBD, and ACT1 to confirm the rcRBD-integration in these cell lines. B Detection of rcRBD mRNA in knock-in rice suspension cell lines. Total RNA was isolated from wild-type (WT), rcRBD-32, and rcRBD-42 suspension cell lines after 6, 12, and 24 h of culture in sugar-starved (-S) medium. RT-PCR analysis was conducted using specific primers for rcRBD, αAmy3_E1-rcRBD, αAmy8_E1-rcRBD, and ACT1. C, D Detection of rcRBD protein in knock-in rice suspension cells. The secreted protein from culture medium (C) and total soluble protein (D) were extracted from WT, rcRBD-32, and rcRBD-42 cells after 8 days of sugar starvation. A total of 7.5 μl of rcRBD32 culture medium protein and 30 μl of rcRBD-42 culture medium protein were loaded in (C). The same amount 80 ug of total soluble protein from each cell lines were loaded in (D). Western blot analysis was performed using RBD antiserum. Recombinant SARS-CoV-2 spike RBD protein produced from HEK293 cells served as a positive control. Molecular weight markers (kDa) are shown on the left side of the figure. Coomassie blue was used as loading control

Fig. 4

Profiling of secreted recombinant rcRBD production in knock-in cell line. A–B Suspension cells from rcRBD-32 (A) and rcRBD-42 (B) cell lines were cultured in a 4 mL of sugar-free MS medium. The culture medium was collected from days 4 to 10, and equal amounts of secreted medium protein from each sample were subjected to western blot analysis using RBD antiserum. C–D The rcRBD productivity in the culture medium of rice suspension cultures. The rcRBD protein levels in rcRBD-32 (C) and rcRBD-42 (D) cell lines were determined by ELISA, relative to a standard using a serial dilution of RBD protein derived from HEK293 cells. Error bars represents the standard deviation (SD) from triplicate cultures

Fig. 5

Antigen–antibody recognition of rice cell secreted rcRBD. The culture medium was collected on day 8 from sugar-starved WT, rcRBD-32, and rcRBD-42 suspension cells. An equal total volume (100 uL) of cultured medium from each cell lines was used to test antigen–antibody recognition using the COVID-19 Antigen Detection Kit (Celltrion DiaTrust™, Humasis Co, Gyeonggi-do, Republic of Korea). A positive result is indicated by the presence of two lines, one at the control (C) region and one at the test (T) region. A negative result is indicated by a single line at the C region