Prime Editing in the model plant Physcomitrium patens and its potential in the tetraploid potato.

Since its discovery and first applications for genome editing in plants, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology has revolutionized plant research and precision crop breeding. Although the classical CRISPR-Cas9 system is a highly efficient tool for disruptive targeted mutagenesis, this system is mostly inefficient for the introduction of precise and predictable nucleotide substitutions. Recently, Prime Editing technology has been developed, allowing the simultaneous generation of nucleotide transitions and transversions but also short defined indels. In this study, we report on the successful use of Prime Editing in two plants of interest: the plant model Physcomitrium patens and the tetraploid and highly heterozygous potato (Solanum tuberosum). In both cases editing rates were lower than with other CRISPR-Cas9 based techniques, but we were able to successfully introduce nucleotide transversions into targeted genes, a unique feature of Prime Editing. Additionally, the analysis of potential off-target mutation sites in P. patens suggested very high targeting fidelity in this organism. The present work paves the way for the use Prime Editing in Physcomitrium patens and potato, however highlighting the limitations that need to be overcome for more efficient precision plant breeding.

Gene Editing in Potato Using CRISPR-Cas9 Technology.

Genome editing in the cultivated potato (Solanum tuberosum), a vegetatively propagated and highly heterozygous species, constitutes a promising trail to directly improve traits into elite cultivars. With the recent and successful development of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system in eukaryotic cells, the plant science community has gained access to a powerful, inexpensive, and easy-to-use toolbox to target and inactivate/modify specific genes. The specificity and versatility of the CRISPR-Cas9 system rely on a variable 20 bp spacer sequence at the 5' end of a single-guide RNA (sgRNA), which directs the SpCas9 (Streptococcus pyogenes) nuclease to cut the target DNA at a precise locus with no or low off-target events. Using this system, we and other teams were able to knock out specific genes in potato through the error-prone non-homologous end-joining (NHEJ) DNA repair mechanism. In this chapter, we describe strategies to design and clone spacer sequences into CRISPR-SpCas9 plasmids. We show how these constructs can be used for Agrobacterium-mediated stable transformation or transient transfection of protoplasts, and we describe the optimization of these two delivery methods, as well as of the plant regeneration processes. Finally, the molecular screening and characterization of edited potato plants are also described, mainly relying on PCR-based methods such as high-resolution melt (HRM) analysis.

Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops.

Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.

The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato.

KEY MESSAGE: The StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base-editing strategies, leading to plants with impaired amylose biosynthesis. Genome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant-breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knock out the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss of function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.

Pyramiding resistances based on translation initiation factors in Arabidopsis is impaired by male gametophyte lethality.

In eukaryotes, eIF4E translation initiation factors are essential proteins encoded by a small multigene family. In plants, they are a major source of host plant resistance to potyviruses that require specific 4E factors to infect cells. Combining mutations in different eIF4E genes could be a way of broadening the spectrum of plant resistance to viruses. We attempted to combine null mutations affecting the two main Arabidopsis thaliana 4E factors eIF4E1 and eIFiso4E but discovered that this combination is lethal. Transmission through the male gametophyte is completely abolished in the eif4e1 eifiso4e double mutant. This shows that eIF4E1 and eIFiso4E are essential for male gametophyte development and act redundantly. These results may have implications for eIF4E-based pyramiding strategies to improve crop resistance.

Functional characterization of the plant ubiquitin regulatory X (UBX) domain-containing protein AtPUX7 in Arabidopsis thaliana.

p97/CDC48 is a major AAA-ATPase that acts in many cellular events such as ubiquitin-dependent degradation and membrane fusion. Its specificity depends on a set of adaptor proteins, most of them containing the ubiquitin regulatory X (UBX) domain. Using a differential hybridization system, we isolated a UBX-containing protein that is expressed during the early phase of male gametophyte development in the crop Brassica napus and isolated and characterized its closest Arabidopsis thaliana homolog, AtPUX7. The AtPUX7 gene is expressed broadly in both the sporophyte and gametophyte due to regulation inferred by its first intron. The subcellular localization of AtPUX7 was assigned mainly to the nucleus in both the sporophyte and in pollen, mirroring the AAA-ATPase AtCDC48A localization. Furthermore, AtPUX7 interacts specifically with AtCDC48A in yeast as well as in planta in the nucleus. This interaction was mediated through the AtPUX7 UBX domain, which is located at the protein C-terminus, while an N-terminal UBA domain mediated its interaction with ubiquitin. Consistent with those results, a yeast-three hybrid analysis showed that AtPUX7 can act as a bridge between AtCDC48A and ubiquitin, suggesting a role in targeted protein degradation. It is likely that AtPUX7 acts redundantly with other members of the Arabidopsis PUX family because a null Atpux7-1 mutant does not display obvious developmental defects.

The Arabidopsis proteasome RPT5 subunits are essential for gametophyte development and show accession-dependent redundancy.

We investigated the role of the ubiquitin proteasome system (UPS), which allows proteins to be selectively degraded, during gametophyte development in Arabidopsis thaliana. Three mutant alleles altering the UPS were isolated in the Wassilewskija (Ws) accession: they affect the Regulatory Particle 5a (RPT5a) gene, which (along with RPT5b) encodes one of the six AAA-ATPases of the proteasome regulatory particle. In the heterozygous state, all three mutant alleles displayed 50% pollen lethality, suggesting that RPT5a is essential for male gametophyte development. However, a fourth mutant in the Columbia (Col) accession did not display such a phenotype because the RPT5b Col allele complements the rpt5a defect in the male gametophyte, whereas the RPT5b Ws allele does not. Double rpt5a rpt5b mutants showed a complete male and female gametophyte lethal phenotype in a Col background, indicating that RPT5 subunits are essential for both gametophytic phases. Mitotic divisions were affected in double mutant gametophytes correlating with an absence of the proteasome-dependent cyclinA3 degradation. Finally, we show that RPT5b expression is highly increased when proteasome functioning is defective, allowing complementation of the rpt5a mutation. In conclusion, RPT5 subunits are not only essential for both male and female gametophyte development but also display accession-dependent redundancy and are crucial in cell cycle progression.

AtPRD1 is required for meiotic double strand break formation in Arabidopsis thaliana.

The initiation of meiotic recombination by the formation of DNA double-strand breaks (DSBs) catalysed by the Spo11 protein is strongly evolutionary conserved. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation, but, unlike Spo11, few of these proteins seem to be conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we have isolated a new gene, AtPRD1, whose mutation affects meiosis in Arabidopsis thaliana. In Atprd1 mutants, meiotic recombination rates fall dramatically, early recombination markers (e.g., DMC1 foci) are absent, but meiosis progresses until achiasmatic univalents are formed. Besides, Atprd1 mutants suppress DSB repair defects of a large range of meiotic mutants, showing that AtPRD1 is involved in meiotic recombination and is required for meiotic DSB formation. Furthermore, we showed that AtPRD1 and AtSPO11-1 interact in a yeast two-hybrid assay, suggesting that AtPRD1 could be a partner of AtSPO11-1. Moreover, our study reveals similarity between AtPRD1 and the mammalian protein Mei1, suggesting that AtPRD1 could be a Mei1 functional homologue.