Genome editing tools have rapidly been adopted by plant scientists for gene function discovery and crop improvement. The current technical challenge is to efficiently induce precise and predictable ...targeted point mutations valuable for crop breeding purposes. Cytidine base editors (CBEs) are CRISPR/Cas9 derived tools recently developed to direct a C-to-T base conversion. Stable genomic integration of CRISPR/Cas9 components through
-mediated transformation is the most widely used approach in dicotyledonous plants. However, elimination of foreign DNA may be difficult to achieve, especially in vegetatively propagated plants. In this study, we targeted the
(
) gene in tomato and potato by a CBE using
-mediated transformation. We successfully and efficiently edited the targeted cytidine bases, leading to chlorsulfuron-resistant plants with precise base edition efficiency up to 71% in tomato. More importantly, we produced 12.9% and 10% edited but transgene-free plants in the first generation in tomato and potato, respectively. Such an approach is expected to decrease deleterious effects due to the random integration of transgene(s) into the host genome. Our successful approach opens up new perspectives for genome engineering by the co-edition of the
with other gene(s), leading to transgene-free plants harboring new traits of interest.
Genome editing is now widely used in plant science for both basic research and molecular crop breeding. The clustered regularly interspaced short palindromic repeats (CRISPR) technology, through its ...precision, high efficiency and versatility, allows for editing of many sites in plant genomes. This system has been highly successful to produce knock-out mutants through the introduction of frameshift mutations due to error-prone repair pathways. Nevertheless, recent new CRISPR-based technologies such as base editing and prime editing can generate precise and on demand nucleotide conversion, allowing for fine-tuning of protein function and generating gain-of-function mutants. However, genome editing through CRISPR systems still have some drawbacks and limitations, such as the PAM restriction and the need for more diversity in CRISPR tools to mediate different simultaneous catalytic activities. In this study, we successfully used the CRISPR-Cas9 system from Staphylococcus aureus (SaCas9) for the introduction of frameshift mutations in the tetraploid genome of the cultivated potato (Solanum tuberosum). We also developed a S. aureus-cytosine base editor that mediate nucleotide conversions, allowing for precise modification of specific residues or regulatory elements in potato. Our proof-of-concept in potato expand the plant dicot CRISPR toolbox for biotechnology and precision breeding applications.
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.
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
and two
crops (
and
) 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 large French research project GENIUS (2012–2019,
https://www6.inra.genius-project_eng/
) provides a good showcase of current genome editing techniques applied to crop plants. It addresses a large ...variety of agricultural species (rice, wheat, maize, tomato, potato, oilseed rape, poplar, apple and rose) together with some models (Arabidopsis, Brachypodium, Physcomitrella). Using targeted mutagenesis as its work horse, the project is limited to proof of concept under confined conditions. It mainly covers traits linked to crop culture, such as disease resistance to viruses and fungi, flowering time, plant architecture, tolerance to salinity and plant reproduction but also addresses traits improving the quality of agricultural products for industrial purposes. Examples include virus resistant tomato, early flowering apple and low-amylose starch potato. The wide range of traits illustrates the potential of genome editing towards a more sustainable agriculture through the reduction of pesticides and to the emergence of innovative bio-economy sectors based on custom tailored quality traits.
•Gene editing by Prime Editing is effective in Physcomitrium patens and Solanum tuberosum.•In Physcomitrium patens, the Prime Editing frequency and its accuracy vary with the target sequence coded by ...the pegRNA.•In Physcomitrium patens, Prime Editing does not appear to generate off-target event at homeologous sites with four or less base mismatches.
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.
The cutaneous microangiopathy plays a key role in the development of the skin clinical lesions of venous insufficiency. Capillaroscopy allows a non-invasive observation of the superficial skin ...capillaries of the lower leg, which have previously been shown to be altered in patients with advanced venous disease. As it is now available in a friendly, easy to handle way through modern video devices, we report our findings in a short series of patients with C3-C5 chronic venous disorders using this technique.
A total of 21 patients with venous insufficiency (C3-C5 on at least one leg) underwent a capillaroscopic examination of both legs and pictures recorded from the sites of the most severe venous skin lesions. This was performed with a CapXview handheld video-capillaroscope (×100 magnification), allowing easy manual measurement of maximum capillary bulk diameter and capillary density.
Dramatic changes in capillary density, size, and shape were easily observed at the site of the venous skin lesions. A significant negative linear relationship was found between capillary density and the "C" classes (
= -0.45;
< 0.001). A significant negative correlation was also found between capillary density and bulk diameter (
= -0.52;
< 0.001). The area under the ROC curve for the mathematical prediction of venous skin changes by capillary density was 0.842, which shows the strength of the link between the microvascular and the clinical status.
Video-capillaroscopy allows a direct observation of the cutaneous venous microangiopathy and provides the possibility to measure capillary density which allows its quantification. This simple to use technique shows the potential for a more precise follow-up and treatment evaluation of the cutaneous consequences of venous disease, which remains to be further investigated.
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.