This document discusses genome editing in fruit crops using CRISPR/Cas9 technology. It provides examples of using CRISPR to edit genes involved in fruit ripening, pigmentation, and flowering time regulation in strawberry, banana, apple, and kiwifruit. Specifically, it describes using CRISPR to increase beta-carotene levels in banana, induce early flowering in apple and pear, and generate dwarf kiwifruit plants. The document concludes that integrating biotechnology like CRISPR with conventional breeding is a promising strategy for fruit crop improvement.

1. M K SAINI
Presented by:
Mandeep Kaur
L-2018-A-35-D
PhD Fruit Science
Presented to:
Dr H S Dhaliwal
Dr Monika Gupta
Dr Rachna Arora
Genome Editing in fruit crops

2. M K SAINI
Genome Editing
• Genome editing is a type of genetic engineering in which DNA
is inserted, deleted, modified or replaced in the genome of a
living organism.
• To alter plant genomes and produce desirable traits without
introducing foreign genes.
• Zinc finger nucleases (ZFNs) and transcription activator-like
effector nucleases (TALENs) are early gene editing systems
that have limited utility because they are complex, time-
consuming, expensive, and inefficient.

3. M K SAINI
CRISPR
• Clustered Regularly Interspaced Short Palindromic Repeat.
• CRISPR technology is easy to use, efficient, and low-cost.
• The CRISPR/Cas9 system is an adaptive immune mechanism in bacteria and
archaea that pro- vides protection against viruses and plasmids.
• The artificially modified CRISPR/Cas9 system consists of two components: a single
guide RNA (sgRNA) that targets a specific DNA sequence with its 5 _ end and the
DNA endonuclease Cas protein that cleaves the target sequence between the third
and fourth nucleotides upstream of the protospacer adjacent motif (PAM).
• Once the Cas protein cleaves the genomic DNA, a double-strand break (DSB)
forms, triggering cellular DNA repair mechanisms known as non-homologous end
joining (NHEJ) and homology-directed repair (HDR).
• The NHEJ pathway is activated quickly and inaccurately with- out a homologous
repair template and introduces small inser- tions/deletions (InDel) or substitutions,
even knocking out the functional gene, whereas the HDR pathway is initiated
precisely using homologous sequences, leading to the insertion of a new sequence
or the replacement of a DNA sequence.
3

4. M K SAINI

5. M K SAINI
CRISPR/Cas9 directed editing of lycopene
epsilon-cyclase (LCYε) gene in banana fruit
• Parthenocarpic nature & triploid genome => Major breeding challenges.
• The fifth exon of LCYε gene was targeted.
• β-carotene content increased to 6-fold (~24 μg/g) in edited lines.
Carotenoid estimation in ripe fruit pulp tissue of the mutant lines
Control
qRT-PCR analysis of β-LCY
Kaur et al (2020) NABI Mohali
GN - 9

6. M K SAINI
CRISPR/Cas9 directed editing of terminal
flower 1 (TFL1) gene in apple and pear
• Long reproductive cycle & high degree of heterozygosity – Breeding problem.
• TFL1 gene is a floral repressor.
• CRISPR-TFL1.1 vector
• MdTFL1.1 gene - Apple cv. Gala
• PcTFL1.1 gene - Pear cv. conference
• TRANSIENT TRANSFORMATION- CRISPR-PDS construct produced two T-DNA free
edited apple lines.
In apple, opened flower with all floral organs present Opened flower in pear
Charrier et al (2019) France

7. M K SAINI
Explant Traits related to fruit quality Reference Mode of
transformation
Explant Traits related to fruit
quality
Reference
FRUIT RIPENING
Fragaria vesca TAA1 , ARF8 CRISPR/Cas9 Agrobacterium Young
cotyledon
Auxin biosynthesis and
signaling
Zhou et al.,
2018
Fragaria vesca FveYUC10 CRISPR/Cas9 Agrobacterium Leaf Reduction of free auxin Feng et al.,
2019
INCREASED PIGMENT CONTENT
Fragaria ananassa MYB10 , CHS CRISPR/Cas9 Agrobacterium Leaf Anthocyanin biosynthesis
decreased
Xing et al.,
2018
Fragaria
ananassa
RAP CRISPR/Cas9 Agrobacterium Leaf Anthocyanin biosynthesis
decreased
Gao et al.,
2020
Fragaria vesca uORF of FvebZIPs1.1 CRISPR/Cas9
Base editor
A3A-PBE
Agrobacterium Leaf Increased sugar content;
transgene-free mutants
contain a continuous
sugar content.
Xing et al.,
2020
REGULATION OF FLOWERING TIME
Malus domestica Phytoene Desaturase (PDS),
Terminal Flower 1 (TFL1)
CRISPR/Cas9 Agrobacterium Leaf Albino phenotype and
early flowering
Charrier et al.,
2019
Pyrus communis TFL1.1 CRISPR/Cas9 Agrobacterium Leaf Early flowering Charrier et al.,
2019
Actinidia chinensis CEN4 , CEN CRISPR/Cas9 Agrobacterium Leaf Generation of a compact
plant with rapid terminal
flower and fruit;
development precocity
Varkonyi-
Gasic et al.,
2019
CRISPR directed genome editing in fruit crops

8. M K SAINI
Conclusion & Future Prospects
• Biotechnology has brought great opportunities and
prospects for overcoming problems of conventional
breeding.
• However, biotechnology as transgenic breeding or genetic
manipulation cannot replace conventional breeding but it is
and only is a supplementary addition to conventional
breeding.
• Therefore, integration of biotechnology into conventional
breeding programs will be an optimistic strategy for fruit
crop improvement in the future.

9. M K SAINI