Engineering herbicide resistance via prime editing in rice

Although CRISPR-Cas9 has revolutionized our ability to generate site-specific double-strand breaks, precise editing of the genome remains challenging in most eukaryotes, including plants (Shan et al., 2013). In plants homology-directed repair is inefficient, limiting our ability to make precise edits of the DNA sequence (Ali et al., 2020; Butt et al., 2017). Moreover, cytosine and adenine base editors have serious drawbacks including lower efficiency, unclean edited sequence, and the possibility of off-target mutations at other loci (Rees and Liu, 2018). Chimeric single guide RNAs (sgRNAs) can provide editing information, in RNA form, but this modality suffers from several limitations including lower efficiency, less versatility, and the need for long homology arms (Butt et al., 2017).

Although CRISPR-Cas9 has revolutionized our ability to generate site-specific double-strand breaks, precise editing of the genome remains challenging in most eukaryotes, including plants (Shan et al., 2013). In plants, homology-directed repair is inefficient, limiting our ability to make precise edits of the DNA sequence (Ali et al., 2020;Butt et al., 2017). Moreover, cytosine and adenine base editors have serious drawbacks including lower efficiency, unclean edited sequence and the possibility of off-target mutations at other loci (Rees and Liu, 2018). Chimeric single-guide RNAs (sgRNAs) can provide editing information, in RNA form, but this modality suffers from several limitations including lower efficiency, less versatility and the need for long homology arms (Butt et al., 2017).
In contrast to genome editing methods that use just a Cas nuclease to generate double-strand breaks, prime editing employs a Cas9 nickase (nCas9) fused with reverse transcriptase (RT). The desired edits are encoded on a prime editing guide RNA, which guides the nCas9-RT complex to the target site (Anzalone et al., 2019). There, the nCas9 generates a single-strand break (Shrivastav et al., 2008) on the non-complimentary strand and the RT domain transfers the desired edits from the pegRNA to the DNA (Anzalone et al., 2019). Researchers have developed several prime editing strategies: in PE1, wild type M-MLV RT fused to the C terminus of Cas9 (H840A) nickase; in PE2, Cas9 (H840A) with pentamutant M-MLV RT (D200N/ L603W/ T330P/ T306K/ W313F); in PE3, a PE2 prime editor with additional simple gRNA to simultaneously nick the non-edited strand (Anzalone et al., 2019). Prime editing has several advantages over other methods, such as enabling precise sequence deletion, addition and substitution. However, although it has been tested in human cell lines, prime editing remains to be tested in plants.
To test prime editing in rice (Oryza sativa), we first attempted to engineer herbicide resistance by targeting rice ACETOLACTATE SYNTHASE (OsALS). ALS catalyses the initial step common to the biosynthesis of the branched-chain amino acids and is primary target site for herbicides like Bispyribac sodium. A single amino acid change (W548L) in ALS results in a BS-resistant phenotype (Butt et al., 2017). We cloned the PE2 fragment containing Cas9 (H840A) with pentamutant M-MLV RT under the control of the OsUBIQUITIN promoter in rice vectors. We therefore designed a pegRNA to edit the OsALS sequence. The RT template with a length of 15 bp has two substitutions, a G-to-T substitution that converts tryptophan 548 to leucine and a silent G-to-A substitution that destroys the PAM site thus preventing re-targeting by the pegRNA-nCas9-RT machinery ( Figure 1a). These nucleotide modifications result in the loss of a BsaXI site and generation of an MfeI site. The primer binding site (PBS) was designed with a length of 13 bp. The pegRNA was expressed in rice vectors under the OsU3 promoter.
We transformed rice via Agrobacterium and after two weeks of selection, we collected four independently growing calli from different selection plates. We performed the DNA extraction from these calli and amplified the target DNA by PCR. We pooled the amplicons in equimolar concentrations and performed deep sequencing. Our data showed that the prime editing successfully edited OsALS at the target site with an efficiency of 0.26 to 2% (Figure 1b). The different editing efficiencies among two pools are possibly due to varied number of non-edited WT cells between these calli. The editing efficiencies are further validated when we enriched the edited DNA from the four calli by cutting with BsaXI (which cuts the unedited sequence) and conducted PCR/restriction enzyme analysis (PCR/RE) using MfeI (Figure 1c). The digestion of amplicons by MfeI indicated the frequency of editing in the samples. We used Sanger sequencing to confirm these edits (Figure 1d). Most of the reads were fully edited and repaired according to the RT template. Interestingly, some of the reads showed an A-to-G substitution, which converts tyrosine 553 to cysteine. This substitution is not the part of the RT template and probably came from the scaffold RNA, as the first nucleotide of the scaffold RNA adjacent to the RT template (a 'G') can be used for DNA repair (Figure 1d).
We also targeted rice IDEAL PLANT ARCHITECTURE 1 (OsIPA) using prime editing (Figure 1e). The OsIPA transcription factor reduces the number of unproductive tillers and improves rice yield. We designed a pegRNA for two consecutive substitutions (AG to GA) to convert S163 to D in IPA with length of RT 20 bp and PBS 13 bp. Two silent substitutions (CGC to AGA) destroy the PAM site. These mutations destroy a PvuII site and generate Pst1 and BbsI sites. We transformed rice via Agrobacterium and regenerated shoots. We analysed the plantlets after enriching for edited DNA with PvuII digestion by Sanger sequencing. We found that prime editing successfully edited OsIPA at the target site, (Figure 1f).
Similarly, we targeted rice TEOSINTE BRANCHED 1 (OsTB1), a member of the TEOSINTE BRANCHED1, CYCLOIDEA AND PCF TRANSCRIPTION FACTOR gene family (Figure 1g). OsTB1 negatively regulates lateral branching by repressing axillary bud outgrowth. We designed a pegRNA to target the OsTB1 promoter with length of RT 20 bp and PBS 13 bp. A C-to-G substitution destroyed the PAM to prevent re-targeting and two consecutive insertions (AA) and one substitution (C to T) destroyed and RsaI site and created an SspI site. We analysed the shoots by enriching the DNA with RsaI digestion and by Sanger sequencing and observed partial repair and different types of reads (Figure 1h). The possible reason for chimeric cells is that prime editing  The amplicons from different rice calli were enriched for editing by BsaXI and after purification, PCR was done and amplicons were digested with MfeI. The digested production indicates the editing in the cells and further confirmed via Sanger sequencing (d). Some of the edited reads, indicated by arrow, were also showing A to G substitutions. This G probably corresponds to the first base of the scaffold RNA adjacent to RT template in pegRNA. (e) Schematic of rice locus IPA (Ideal Plant Architecture). We have designed a pegRNA for two consecutive substitutions AG to GA to convert S163 to D. Two silent substitutions have been done CGC to AGA which convert R165 to R and destroy the PAM site. By these mutations, PvUII site was lost and two sites Pst1 and BbsI were generated. (f) The PvUII enriched DNA samples are confirmed by Sanger sequencing.
(g) Schematic of rice locus TB1 (TEOSINTE BRANCHED1). We have designed a pegRNA to target the GTAC motif in promoter of OsTB1. In the repair, template C was converted to G to destroy PAM site. Two consecutive insertions AA and one substitution were done to destroy binding motif. These mutations also created SspI restriction site and destroy RsaI site. Engineering herbicide resistance via prime editing 2371 machinery could be still functional in the non-edited cells and continuously modified the targeted region.
To test whether we could improve the editing efficiency, we tried the PE3 strategy, where a second sgRNA is used to nick the complimentary strand. We designed the sgRNA to target OsALS at a distance of +55 from the pegRNA and expressed this sgRNA from the OsU3 promoter using the polycistronic tRNA-gRNA system (Butt et al., 2017;Xie et al., 2015) (Figure 1a). We transformed the rice callus with ALS-PE2 (containing just the pegRNA and RT-nCas) and ALS-PE3 (containing the sgRNA, pegRNA, and RT-nCas) plasmids. After selection, we regenerated shoots on media supplemented with 0.75 µM BS (Figure 1i). For both ALS-PE2 and ALS-PE3, we recovered shoots resistant to BS. Sanger sequencing showed that these plantlets were successfully edited (Figure 1j). We recovered almost equal numbers of shoots from PE2 and PE3 (Figure 1k), suggesting that (unlike mammalian systems) PE3 did not increase editing efficiency in plants.
In the present study, we successfully used prime editing technology on three loci in plants. While this work was prepared for publication, similar findings were reported in pants Lin et al., 2020;Tang et al., 2020). We engineered herbicide resistance trait in rice via nucleotide substitutions; however, the system requires further improvements and assessments on its ability to enable diverse editing modalities for different trait engineering applications in plants.