Recently developed CRISPR-mediated base editors, which enable the generation of numerous nucleotide changes in target genomic regions, have been widely adopted for gene correction and generation of crop germplasms containing important gain-of-function genetic variations. However, to engineer target genes with unknown functional SNPs remains challenging. To address this issue, we present here a base-editing-mediated gene evolution (BEMGE) method, employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA (sgRNA) library tiling the full-length coding region, for developing novel rice germplasms with mutations in any endogenous gene. To this end, OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bombardment-mediated transformation. Four different types of amino acid substitutions in the evolved OsALS1, derived from two sites that have never been targeted by natural or human selection during rice domestication, were identified, conferring varying levels of tolerance to the herbicide bispyribac-sodium. Furthermore, the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing with the corresponding base editor and sgRNA. Collectively, these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.

Background Plant genome engineering mediated by various CRISPR-based tools requires specific protospacer adjacent motifs (PAMs), such as the well-performed NGG, NG, and NNG, to initiate target recognition, which notably restricts the editable range of the plant genome. Results In this study, we thoroughly investigate the nuclease activity and the PAM preference of two structurally engineered SpCas9 variants, SpG and SpRY, in transgenic rice. Our study shows that SpG nuclease favors NGD PAMs, albeit less efficiently than the previously described SpCas9-NG, and that SpRY nuclease achieves efficient editing across a wide range of genomic loci, exhibiting a preference of NGD as well as NAN PAMs. Furthermore, SpRY-fused cytidine deaminase hAID*Δ and adenosine deaminase TadA8e are generated, respectively. These constructs efficiently induce C-to-T and A-to-G conversions in the target genes toward various non-canonical PAMs, including non-G PAMs. Remarkably, high-frequency self-editing events (indels and DNA fragments deletion) in the integrated T-DNA fragments as a result of the nuclease activity of SpRY are observed, whereas the self-editing of SpRY nickase-mediated base editor is quite low in transgenic rice lines. Conclusions The broad PAM compatibility of SpRY greatly expands the targeting scope of CRISPR-based tools in plant genome engineering.

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Peru is a centre of origin and domestication of the potato, pepper and tomato (family Solanaceae). Many potyviruses (genus Potyvirus) that infect these crops were described 20-30 years ago. However, definitive classification of these viruses as distinct species remains unresolved for several reasons, including their close serological relationships, similar symptomatology in test plants and lack of genomic sequence data. Using samples collected from Peru, we have determined the complete genomic sequence of two strains of Peru tomato virus (PTV) as well as near-complete sequences for two additional PTV strains. We also obtained partial sequences of four strains of Potato virus V (PVV). Comparisons with genomic sequences of Wild potato mosaic virus (WPMV), Potato virus Y (PVY), Pepper mottle virus (PepMoV), Potato virus A (PVA) and other potyviruses established that all these viruses constitute different taxa (species). Phylogenetic comparisons indicated that PTV, PVV and WPMV are the most closely related species which, together with PepMoV, PVY, Pepper yellow mosaic virus and Pepper severe mosaic virus, constitute a group that is distinguishable from other potyviruses. Therefore, the members of this group may share a common ancestor. PVA does not belong to this group. PVV and PTV were also closely related serologically. However, PTV did not cross-protect against PVV and WPMV in tobacco plants or complement systemic infection of PVV and WPMV in pepper plants. Two biologically and phylogenetically distinguishable strain groups were identified within PTV and PVV. In future studies, the sequence data and virus-specific primers and probes for PTV, PVV and WPMV described in this study will enable accurate indexing of plants with respect to either single or mixed infection with these viruses.

Because there were no previous reports on the molecular characterization of Potato virus V (PVV, genus Potyvirus, family Potyviridae), the complete genomic sequence of PVV isolate Dv42 was determined. The length of the single-stranded messenger-polarity RNA genome was 9,851 nt (nucleotides), followed by a poly(A) tail. The genome contained a 5'-terminal nontranslated region (5'-NTR; 204 nt), a single open reading frame (nucleotides 205-9406; 3,067 amino acids), and a 3'-NTR that was unusually long (446 nt) compared with that of Potato virus Y (PVY; 331-nt 3'-NTR), Potato virus A (PVA; 207-nt 3'-NTR), and other potyviruses that naturally infect Solanaceae species. Phylogenetic analysis with the cylindrical inclusion protein-encoding and coat protein (CP)-encoding regions indicated that PVV Dv42 was most closely related to Pepper mottle virus and PVY, respectively. Seven PVV isolates (including Dv42) collected from cultivated potatoes in the Netherlands, the United Kingdom, and Norway from 1964 to 1997 were uniform in serological properties and symptomatology in indicator hosts that could distinguish strains of PVY and PVA. The nucleotide sequences of the 5'-NTR, P1, CP, and 3'-NTR regions of the PVV isolates were determined and were 94.6 to 99.5, 96.3 to 98.8, 96.4 to 98.7, and 96.3 to 99.6% identical, respectively. The amino acid similarities for the P1 and CP were 95.8 to 98.6 and 96.0 to 97.8%, respectively. Phylogenetic analysis of the CP sequences of PVV revealed no significant grouping, in contrast to PVY and PVA, which were grouped largely according to the previously recognized strains based on host responses. However, the relatively few differences in the P1 sequences of PVV were correlated with the different countries of origin. Hence, the PVV isolates infecting potatoes in Europe seem to vary little genetically and may belong to a single strain.