CRISPR/CAS9 EDITING OF THE

POLYGALACTURONASE FAPG1
GENE IMPROVES STRAWBERRY
FRUIT FIRMNESS

SEBASTIAN ALVAREZ ATEHORTUA

UNIVERSITY OF ANTIOQUIA
2023
THE CULTIVATED STRAWBERRY (FRAGARIA ×
ANANASSA, DUCH.)

• It is one of the most popular species of

the Rosaceae family. The organoleptic
properties of strawberries make this
fruit one of the most important crops in
the world.
• The soft texture of strawberries
determines their short postharvest life
and causes important economic losses.
economic losses
FACTORS INVOLVED IN FRUIT
DEGRADATION

• Cell wall disassembly and

middle lamella dissolution
are the main causes of fruit
softening.
 Polygalacturonases (PGs) β-galactosidases Pectate lyases

Functional analyses have shown that pectinase enzymes play a key role
in fruit softening.
• Transgenic silencing of
some of these specific
genes reduced softening
and increased postharvest
shelf life.
• It has been reported that
with the PG gene of FaPG1,
silencing by antisense
transformation significantly
increased fruit firmness.
 CRISPR/CAS9 APPLICATION

The PG gene was described in strawberry, being

FaPG1, whose silencing by antisense transformation
significantly increased fruit firmness.

CRISPR/Cas9 has been used to edit the endogenous

marker gene phytoene desaturase.
Genomic DNA of AF380299.1
Lanes showing a double band indicate successful gene editing.
 TRANSFORMATION AND REGENERATION
OF FAPG1 EDITED PLANTS
• The sequence was used to select an
sgRNA to induce single mutations in the
gene.

• To determine any allelic variation that

might affect CRISPR/Cas9 editing, FaPG1
was amplified from mature plant cDNA.
 PHENOTYPIC ANALYSES OF TRANSGENIC
PLANTS
• The primary transgenic lines were
acclimatized to greenhouse conditions
and propagated by stolons

• Fruits of eight selected lines were

harvested at the red-ripe stage, with the
entire fruit surface reddened, and quality
parameters were analyzed
• In this postharvest experiment, it
was observed that the incidence of
fungal rot symptoms at seven days
postharvest was lower in edited
fruit compared to controls.
Botrytis cinerea strain B05.10
RESULTS

• The polygalacturonase gene FaPG1 was successfully edited in cultured

strawberry using the CRISPR/Cas9 system released by A. tumefaciens.
• FaPG1 editing was not only beneficial in reducing the rate of softening
in strawberry, but also in reducing fruit susceptibility to Botrytis cinerea
infection and in reducing transpirational water loss.
BIBLIOGRAPHY
• -López-Casado, G. et al. (2023) ‘CRISPR/Cas9 editing of the polygalacturonasefapg1gene improves strawberry
fruit firmness’, Horticulture Research, 10(3). doi:10.1093/hr/uhad011.

• The strawberry (Fragaria×ananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an

enzyme involved in the degradation of cell-wall middle lamellae - Scientific Figure on ResearchGate. Available
from: https://www.researchgate.net/figure/Ultrastructural-images-of-cell-walls-in-control-and-FaGLyase1-
silenced-strawberry-fruit_fig8_236129559 [accessed 8 Jul, 2023]

• Posé, S. et al. (2019) ‘A nanostructural view of the cell wall disassembly process during fruit ripening and
postharvest storage by atomic force microscopy’, Trends in Food Science & Technology, 87, pp. 47–58.
doi:10.1016/j.tifs.2018.02.011.
THANK YOU