Precise gene editing in rice using paired guides targeting NG or NGG PAM sites

The paired epegRNAs and 8-nt linkers between PBS and tevopreQ1 for epegRNAs were designed with pegFinder (http://pegfinder.sidichenlab.org/↗) (Chow et al., 2021) or PlantPegDesinger (http://www.plantgenomeediting.net/↗) (Lin et al., 2021) and pegLIT (https://peglit.liugroup.us/↗) (Nelson et al., 2022), respectively. To construct the epegRNA expression cassettes, oligonucleotide pairs pegRNA-F/R, including a 20-nt complementary sequence at each 5′and 3′end, respectively, were synthesized. Fragments synthesized by overlap extension PCR (Nelson and Fitch, 2011) (the 20-nt pegRNA spacer scaffold sequenceRT templatePBS8-nt linker) were introduced into the BbsI site of pOsU6_BbsIx2_tQ1_polyT (Nishizawa-Yokoi et al., manuscript submitted) using the In-fusion HD cloning system (Takara). Alternatively, complementary oligonucleotides, pegRNA_5′F/5′R and pegRNA_3′F_Lt/3′R_Lt (listed in Supplementary Table S1), which consist of the 20-nt pegRNA spacer5′-half of scaffold sequence and 3′-half of scaffold sequenceRT templatePBS8-nt linker, respectively, were annealed and ligated together into the BbsI site of pOsU6_BbsIx2_tQ1_polyT.

The PE-NG expression vector, pZH/PE-NG (Os Opt), was constructed from the CRISPR/Cas9 expression vector described in a previous study as pPZP_ZmUbi:BlCas9 (Negishi et al., manuscript submitted). The BlCas9 coding sequence was digested with PmeI/KpnI from the pPZP_ZmUbi:BlCas9 vector and replaced with an Oryza sativa codon-optimized SpCas9-NG (R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R) coding sequence (Endo et al., 2019; Nishimasu et al., 2018) using a conventional ligation method. The SpCas9-NG expression vector was digested with AatI/BsrGI, and an N863A mutation was introduced by inserting a SpCas9-N863A fragment amplified by PCR using the primer sets listed in Supplementary Table S1 using the In-fusion HD cloning system (Takara). An O. sativa codon-optimized M-MLV RT coding sequence [SV40 NLS_engineered M-MLV RT (D200N/L603W/T330P/T306K/W313F) (Anzalone et al., 2019)_SV40 NLS] was amplified by PCR and introduced into the KpnI/SacI site located at the C-terminal region of nSpCas9-NG N863A using the In-fusion HD cloning system (Takara). The paired epegRNA expression cassettes were amplified independently by PCR with the primers shown in Supplementary Table S1, and were introduced concomitantly into the AscI/PacI site of pZH/PEwt (Os Opt) (Nishizawa-Yokoi et al., manuscript submitted) or pZH/PE-NG vector using the In-Fusion Snap cloning kit (Takara). These binary vectors were transformed into Agrobacterium tumefaciens EHA105 (Hood et al., 1993) by electroporation (Bio-Rad).

Rice (O. sativa L.) cv. Nipponbare or the high-yield cultivar Yamadawara were used for the editing the OsEPSPS or OsHSL1 gene, respectively. Four-week-old rice calli derived from embryo scutellum were used for Agrobacterium-mediated transformation following the procedure of Toki et al. (2006). After 3 days co-cultivation, transformed calli were washed and transferred to N6D medium containing 50 mg/L hygromycin B (Wako Pure Chemicals) and 25 mg/L meropenem (Wako Pure Chemicals) for 2 weeks. Antibiotic-resistant clonal callus lines were transferred to new N6D medium containing 50 mg/L hygromycin B and 25 mg/L meropenem and cultured for a further week. Transgenic callus lines carrying the desired mutations in the target gene were transferred to regeneration medium containing 25 mg/L meropenem and cultured for 4 weeks for plant regeneration. T₁ progeny plants were obtained from self-pollinating T₀ plants containing desired mutations in the OsEPSPS locus and were subjected to genotyping of the OsEPSPS gene to confirm the existence of the transgene. T₂ progeny plants were obtained from T₁ plants carrying the homozygous OsEPSPS-PS gene and were subjected to a herbicide-susceptibility test.

Genomic DNA was extracted from clonal propagated transgenic calli according to the protocol for rapid DNA extraction reported by Kasajima et al. (2004). PE frequency in transgenic rice calli was calculated as the ratio of the number of calli carrying the targeted mutation, as detected by direct sequencing of PCR products, to the total number of transgenic calli analyzed.

To detect the targeted PE-induced mutation in the transgenic calli, and to confirm the existence of the desired mutation and undesired scaffold-derived byproducts in regenerated plants, PCR amplification was performed with KOD one PCR master mix (TOYOBO) using the primer sets listed in. The PCR products were purified with AMPure XP (Beckman Coulter) and subjected to sequencing analysis using a Sanger sequencing kit (Thermo Fisher). Supplementary Table S2

Seeds of wild-type rice (Nipponbare) and T₂ progeny plants carrying homozygous OsEPSPS-PS gene were sown on 1/2MS medium with or without glyphosate (NACALAI TESQUE, INC.) and grown in a growth chamber at 27°C under a 16 h photoperiod.

When comparing two data sets, Student's t-test was used and significant differences are indicated with asterisks (*p < 0.05).

To compare PE efficiency using PE-NG with an nSpCas9-NG variant targeting NG-PAM (Nishimasu et al., 2018) and PE-wt with a canonical nSpCas9 targeting NGG-PAM, we designed two pairs of epegRNAs introducing T173I and P177S (TIPS) mutations conferring resistance to the herbicide glyphosate into the rice five-enolpyruvylshikimate-3-phosphate synthase (OsEPSPS) gene (Achary et al., 2020) (Figure 1Ai). Using PE-NG, paired epegRNAs were designed to recognize NG-PAM (NG and CN) adjacent to the editing sites, resulting in nicking at a site adjacent to the target sites with the short available RT template (Figure 1Aii). On the other hand, we used paired epegRNAs targeting NGG-PAM (NGG and CCN) distal from the target site for PE-wt (Figure 1Aiii). A binary vector for PE-NG or PE-wt with paired epegRNAs was transformed into rice calli to test PE efficiency at the OsEPSPS locus. We detected TIPS mutations in OsEPSPS (OsEPSPS-TIPS) in PCR products spanning the target site generated from genomic DNA extracted from transgenic calli. Sequence analysis showed that TIPS mutations were found in 1.9% and 5.6% of transgenic calli carrying PE-NG and PE-wt with paired epegRNAs, respectively (Figures 1B, 2A). Next, we compared PE frequency when introducing the F140H (TTt to CAt) mutation, which confers tolerance to the HPPD herbicide, into the rice HPPD Inhibitor sensitive-like 1 (OsHSL1) gene (Figure 1Ci), between PE-NG and PE-wt (Dong et al., 2024) (Figure 1C(ii), (iii)). The desired modifications were detected by PCR/sequencing analysis in only a few percent and in >20% of callus lines transformed with paired epegRNAs and PE-NG or PE-wt, respectively (Figures 1D, 2B).

We also evaluated PE frequency between PE-NG and PE-wt with paired epegRNAs at another three target sites in rice calli (Supplementary Figures S1A-C). We designed paired epegRNAs for PE-NG and PE-wt to convert C44 to stop (-GC), A256 to T (G to A), and A31 to stop (G to T and -C) in the drooping leaf (OsDL) gene, the suppressor of salicylate insensitivity 2 (OsSSI2) gene, and the phytoene desaturase (OsPDS) gene, respectively (Supplementary Figures S1A-C). The paired epegRNAs for PE-NG recognized NGC-/NGT-, NGC-/NGT-, and NGT-/NGA-PAM in OsDL, OsSSI2, and OsPDS, respectively. OsDL and OsPDS have been used as selective marker genes for genome editing in several plant species (Lin et al., 2021; Mikami et al., 2015), because knockout of these genes has been reported to lead to albino (Qin et al., 2007) and drooping leaf (Yamaguchi et al., 2004) phenotypes, respectively. Substitution of A256T in OsSSI2—one of two amino acid mutations reported as a novel mutant allele of Arabidopsis SSI2 (Yang et al., 2016)—could potentially confer disease resistance on rice. We found that PE-NG (3.82% at OsDL and 0.26% at OsSSI2) was less efficient than PE-wt at the OsDL and OsSSI2 loci (55.21% at OsDL and 1.83% at OsSSI2, Figures 2C,D), while there was no significant difference in frequency between PE-NG and PE-wt at the OsPDS gene (5.90%, PE-NG and 5.21%, PE-wt; Figure 2E).

Transgenic calli with OsEPSPS-TIPS were transferred to regeneration medium to obtain mutant plants harboring the desired mutations; 24 plants were obtained from each of four and five independent transgenic lines expressing PE-NG and PE-wt coupled with paired epegRNAs, respectively, and the genotypes of their OsEPSPS genes were determined by PCR and sequence analysis. Either T173I or P177S mutations (TIPS) or both were detected in at least one plant from all transgenic callus lines transformed with PE-wt (Figure 3A). On the other hand, we found OsEPSPS-TIPS mutant plants from only one line transformed with PE-NG (Figure 3A). In addition, no undesired by products were detected in any regenerated plants expressing either PE-NG or PE-wt (Figures 3A,B). Furthermore, transgenic plants were regenerated from callus lines carrying the PE-modified OsHSL1, OsDL, OsSSI2, and OsPDS genes. Between 7 and 24 plants were obtained from each transgenic callus line expressing PE-NG and PE-wt coupled with paired epegRNAs, and the genotypes of their target genes were determined by PCR and sequence analysis (Table 1; Figures 3C–F). Multiple plants harboring a heterozygous or homozygous F140H mutation in the OsHSL1 gene were observed in all lines transformed with PE-wt (Table 1; Figure 3C). Similarly, in the OsDL, OsSSI2, and OsPDS genes, the proportion of regenerated plants carrying the hetero or homozygous desired mutation derived from callus lines expressing PE-wt was consistently higher than that of regenerated plants expressing PE-NG (Table 1; Figures 3D–F). However, undesired scaffold-derived byproducts in OsHSL1, OsDL, and OsSSI2 were detected concomitantly in several regenerated plants (Table 1; Supplementary Figures S2A-C).

Although we attempted to obtain T₁ progenies from T₀ regenerated plants harboring homozygous OsEPSPS-TIPS gene to test glyphosate sensitivity, no T₁ progenies were obtained. Thus, T₁ progenies with homozygous P177S mutation in the OsEPSPS gene (OsEPSPS-PS) were obtained from regenerated plants with heterozygous OsEPSPS-PS, and their T₂ progenies were subjected to glyphosate sensitivity testing (Supplementary Figure S3A). These plants showed a herbicide glyphosate-tolerant phenotype compared with wild-type plants (Supplementary Figure S3B). Furthermore, PCR analysis confirmed the presence of progeny plants carrying a homozygous OsEPSPS-PS gene and segregating the PE vector (Supplementary Figure S3C).

EPSPS catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate to the 5-hydroxy position of shikimate-3-phosphate in the sikimate pathway, which is an essential pathway for the synthesis of aromatic amino acids (Funke et al., 2009). It has been reported that the EPSPS-TIPS mutant enzyme from Escherichia coli was essentially insensitive to glyphosate, while its activity was decreased significantly compared with wild-type enzyme (Funke et al., 2009). Thus, consistent with a previous report (Li et al., 2016), OsEPSPS-TIPS homozygous mutants were thought to be lethal.