Prime editors, which are CRISPR-Cas9 nickase (H840A)reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%. Introduction of genome modifications such as substitutions, insertions, and deletions that improve agronomic traits can accelerate crop improvement and breeding 1,2. In plants, nuclease-initiated homology-directed repair (HDR) is limited by low efficiency and the difficulty of DNA template delivery 3-6. Cytosine and adenine base editors (CBEs and ABEs) install C•G-to-T•A and A•T-to-G•C transitions 7-9 , and have been successfully used in plants 4. However, base editors are unable to install transversions, insertions, or deletions 7-10. Prime editing uses engineered Cas9 nickase-reverse transcriptase (RT) fusion proteins paired with a pegRNA that encodes the desired edit 11. The RT domain uses a nicked genomic DNA strand as a primer for the synthesis of an edited DNA flap templated by an extension on the pegRNA. Subsequent DNA repair incorporates the edited flap, permanently installing the programmed edit 11. To optimize prime editing for plants, we first compared three plant prime editor systems (PPEs): PPE2, PPE3, and PPE3b 11 (Fig. 1a). PPE2 consists of a nCas9(H840A) fused to an engineered M-MLV RT, and a pegRNA composed of a primer binding site (PBS) and an RT template 11. PPE3 adds an additional nicking single guide RNA (sgRNA) to cleave the non-edited strand, which facilitates favorable DNA repair. In PPE3b, this nicking sgRNA targets the edited sequence, thereby preventing nicking of the non-edited strand until after editing occurs, resulting in fewer indels in mammalian cells 11. We codon-optimized PPE genes for cereal plants and expressed them using the maize Ubiquitin-1 (Ubi-1) promoter (Fig. 1b). We used the OsU3 (or TaU6) and TaU3 promoters to drive pegRNA and nicking sgRNA transcription, respectively. To test whether other RTs support prime editing, we replaced the engineered M-MLV RT with either the CaMV RT (RT-CaMV) from cauliflower mosaic virus 12 or a retron-derived RT (RT-retron) from E. coli BL21 (ref. 13) (Fig. 1b). We first used our previously described 14 rice protoplast reporter system to test the PPE system for blue fluorescent protein (BFP) to green fluorescent protein (GFP) conversion, which requires changing codon 66 from CAC (histidine) to TAC (tyrosine)

Fusarium head blight (FHB), a devastating disease that affects wheat, is caused by a complex of Fusarium species. The overall impact of Fusarium spp. in wheat production arises through the combination of FHB and mycotoxin infection of the grain harvested from infected wheat spikes. Spike infection occurs during opening of flowers and is favoured by high humidity or wet weather accompanied with warm temperatures. Available possibilities for controlling FHB include the use of cultural practices, fungicides and biological approaches. Three cultural practices are expected to be of prime importance in controlling FHB and the production of mycotoxins: soil preparation method (deep tillage), the choice of the preceding crop in the rotation and the selection of appropriate cultivar.

Genetic improvement of grain yield is always an important objective in wheat breeding. Here, a genome-wide association study was conducted to parse the complex genetic composition of yield-related traits of 105 elite wheat varieties (lines) using the Wheat 90K Illumina iSelect SNP array. Nine yield-related traits, including maximum number of shoots per square meter (MSN), effective number of spikes per square meter (ESN), percentage of effective spike (PES), number of kernels per spike (KPS), thousand-kernel weight (TKW), the ratio of kernel length/kernel width (RLW), leaf-area index (LAI), plant height (PH), and grain yield (GY), were evaluated across four environments. Twenty four highly significant marker-trait associations (MTAs) (P < 0.001) were identified for nine yield-related traits on chromosomes 1A, 1D, 2A (2), 3B, 4A (2), 4B, 5A (4), 5B (4), 5D, 6B (2), 7A (2), and 7B (3), explaining 10.86–20.27% of the phenotypic variations. Of these, four major loci were identified in more than three environments, including one locus for RLW (6B), one locus for TKW (7A), and two loci for PH (7B). A cleaved amplified polymorphic sequence (CAPS) marker Td99211 for TKW on chromosome 5A was developed and validated in both a natural population composed of 372 wheat varieties (lines) and a RIL population derived from the cross of Yangxiaomai × Zhongyou 9507. The CAPS marker developed can be directly used for marker-assisted selection in wheat breeding, and the major MTAs identified can provide useful information for fine-mapping of the target genes in future studies.

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