-
Categories
-
Pharmaceutical Intermediates
-
Active Pharmaceutical Ingredients
-
Food Additives
- Industrial Coatings
- Agrochemicals
- Dyes and Pigments
- Surfactant
- Flavors and Fragrances
- Chemical Reagents
- Catalyst and Auxiliary
- Natural Products
- Inorganic Chemistry
-
Organic Chemistry
-
Biochemical Engineering
- Analytical Chemistry
-
Cosmetic Ingredient
- Water Treatment Chemical
-
Pharmaceutical Intermediates
Promotion
ECHEMI Mall
Wholesale
Weekly Price
Exhibition
News
-
Trade Service
CRISPR-Cas9 technology has revolutionized plant research and crop breeding
since it was first applied to plant genome editing.
However, although the classical CRISPR-Cas9 system can target mutagenesis, it is mostly inefficient in nucleotide substitution, and there are off-target effects
.
In 2019, David Liu's lab at the Broad Institute developed Prime Editing technology, which enables precise base "find and replace," adding new tools
to plant breeding.
Recently, researchers at the University of Saclay in Paris, France, successfully applied Prime Editing technology to two plants, small standing moss and potato, paving the way
for future precision crop breeding.
The study was published in the journal Plant Science
.
The Prime Editing system uses modified guide RNA (pegRNA) and prime editor fusion proteins
.
pegRNA has a dual function, both to guide the edited protein to the site of interest and to contain the edited template sequence
.
Prime editor protein is formed by the fusion of Cas9 lipase and reverse transcriptase
.
After Cas9 digestion of the target site, reverse transcriptase performs reverse transcription using pegRNA as a template and then polymerizes the DNA directly onto
the cut DNA strand.
Since its development, Prime Editing technology has been successfully applied to several plants, such as rice, wheat and corn
.
In this study, the researchers wanted to evaluate the potential of this technique on two different plants, including the model organism Physcomitrium patens and the highly heterozygous tetraploid potato (Solanum tuberosum).
To quickly identify the edits, they used a different reporting system
for each plant.
For P.
small, they chose the PpAPT gene, which is able to convert 2-fluoroadenine (2FA) to 2-fluoro-AMP, a lethal compound
that can be used for reverse selection in plants.
Thus, editing of the PpAPT gene will confer the ability of plants to grow on medium containing 2FA (Figure 1).
For potatoes, they chose to modify the acetyl mastogram synthase (ALS) gene to make it resistant to the herbicide chlorsulfuron
.
Figure 1.
Prime Editing strategy for precise modification of the PpAPT gene
Gene editing of Bulbophyllum parspp
The researchers tested two versions of the editing system, PE2 and PE3, which differ in that PE3 adds sgRNA
that can cut non-coding strands.
They found that PE2 was able to function in small standing bowl moss, and 6 out of 8 pegRNAs produced plants
with 2FA resistance.
The coding sequence of the Prime Editing enzyme and the pegRNA-expressing element were synthesized
by Twist Bioscience.
Remarkably, even the best-potent No.
6 pegRNAs were observed at two orders of magnitude lower in mutation frequencies at the PpAPT locus than standard Cas9 or Base Editor (Table 1).
However, the accuracy of the editing proves the specificity
of Prime Editing on the small standing bowl moss.
In addition, PE3, although increasing the shearing of non-coding chains, did not improve the quality and quantity
of the edits obtained.
Table 1.
Efficiency of PE2 and PE3 use in moss
When using Cas9 technology, the specificity of gene editing is a long-standing problem
.
Therefore, the researchers also carried out off-target analysis
this time.
They designed primers to amplify each predicted off-target site and sequenced
the amplicon.
They found that Prime Editing was highly specific in P.
nigrum, and no modifications
were detected at these prediction sites.
Gene editing of potatoes
They then evaluated PE2 and PE3
on the potato cultivars Desiree.
The gene editing targets the StALS1 and StALS2 genes (the pegRNA-stALS and sgRNA-StALS constructs they use are also synthesized by Twist Bioscience).
High-resolution melting curve (HRM) analysis showed that of the two plants regenerated on chlorsulfuron-containing medium, one plant had a similar melting curve profile to the control (it escaped selection pressure), while the other showed a mutation curve
on the target locus.
Subsequent analyses confirmed that PE2 editing achieved the expected base substitution, but that the number of resistant plants grown on chlorsulfuron medium was very small
.
In addition, PE3 failed to achieve editing
of the ALS gene in potatoes.
Is this inefficiency related to the structure of pegRNA? After the analysis, the researchers found that the pegRNA structure did not significantly destroy Cas9 activity, and the reason for the low efficiency may not be poor targeting the target site
.
The researchers concluded that Prime Editing was less
efficient overall than standard Cas9 or Base Editor techniques in small standing moss and potatoes.
Standard Cas9 typically achieves plant mutagenesis efficiencies of 80%, while Prime Editing can achieve a maximum editing efficiency of only 51% (corn).
They argue that differences between expression systems can partially explain these changes
.
By replacing the promoter, it is expected to improve editing efficiency, especially in potatoes
.
They believe that Prime Editing has the advantage of enabling precise editing at the site of interest and no off-target mutagenesis at the predicted off-target site, but it still needs further improvement before it can be widely used in basic research and precision crop breeding
.
This improvement is especially necessary
for vegetative propagation and polyploid plants.
Original search
Perroud PF, Guyon-Debast A, Veillet F, Kermarrec MP, Chauvin L, Chauvin JE, Gallois JL, Nogué F.
Prime Editing in the model plant Physcomitrium patens and its potential in the tetraploid potato.
Plant Sci.
2022 Mar; 316: 111162.
doi: 10.
1016/j.
plantsci.
2021.
111162.
