CRISPR/Cas systems: The link between functional genes and genetic improvement. The discovery and modification of CRISPR/Cas system, a nature-occurred gene editing tool, opens an era for studying gene function and precision crop breeding cutting-edge biotechnological tool for crop improvement Used for pathogen resistance, abiotic tolerance, plant development and morphology and even secondary metabolism and fiber development

1. CRISPR/Cas systems: The link between
functional genes and genetic
improvement
ANANYA
1ST PhD
PAMB0077

2. Introduction
• Ultimate goal of scientists and breeders: to precisely
control a gene for studying its function as well as
improving crop yield, quality, and tolerance to various
environmental stresses
• The discovery and modification of CRISPR/Cas system, a
nature-occurred gene editing tool, opens an era for
studying gene function and precision crop breeding
• cutting-edge biotechnological tool for crop improvement
• Used for pathogen resistance, abiotic tolerance, plant
development and morphology and even secondary
metabolism and fiber development

3. CRISPR- CAS system
 Clustered regularly interspaced short palindromic
repeats
 Cas (CRISPR-associated protein)
 Is an adaptive phage immunity system present in
archaea and bacteria.

4. Classification of CRISPR

5. CRISPR/Cas system working mechanisms
and CRISPR/Cas family
• The commonly used Cas endonucleases, Cas9, contain
two nuclease domains, RuvC and HNH, and a PAM-
interacting domain (PI)
• RuvC and HNH domains cleave the double-strand
DNA and form a double-strand break (DSB)
• The function of RuvC and HNH domains requires the
Cas nuclease to bind a specific DNA location that are
protospacer adjacent motif (PAM) dependent.
• After Cas cuts the DNAs, DSBs will be repaired using
the cell own DNA repair mechanisms.
• There are two repair pathways, one is non-homologous
end joining (NHEJ) repair and another one is
homology-directed repair (HDR)

6. How does Cas nuclease precisely cut a
specific sequence within the genome?
• One is that CRISPR/Cas system needs one gRNA that
contains both crRNA and tracRNA
• the gRNA or sgRNA is usually a short synthetic RNA
containing a scaffold tracRNA sequence and a spacer
with about 20 nucleotides
• crRNA guides the Cas nuclease to the target sequence
• The tracRNA serves as scaffold for Cas enzyme binding
• The spacer is a sequence complementary with the target
site, which is user-designed sequence and commonly
called gRNA when we design the CRISPR/Cas system
for gene editing

7. • gRNA can target either strand of a gene due to the
Cas enzyme have two nuclease domains, one cuts
sense strand and another one cuts the anti-sense
strand
• PAM is required for Cas enzyme function and
PAM sequence serves as a binding signal for Cas
nuclease; without a PAM sequence, Cas enzyme
does not know where to bind and where to cut
the sequence
• Thus, PAM sequence is required for all current
CRISPR/Cas systems used for genome editing.

8. CRISPR/ CAS9 SYSTEM

10. Mechanism
• Double-strand-break (DSB) repair mechanisms
operate in somatic cells to repair damages in nuclear
DNA.
• DSB repair mechanisms:
1. Homologous recombination (HR): DNA templates
bearing sequence similarity to the break site are used to
introduce sequence changes to the target locus
2. Nonhomologous end joining (NHEJ): The broken
chromosomes are rejoined, often imprecisely, thereby
introducing nucleotide changes at the break site

12. • CRISPR/Cas-based genome editing technologies
serves as the bridge between functional genes and
genetic improvement
• These technologies provide rice researchers with
the opportunity to introduce specific and explicit
changes at target loci and can be used for knocking
out multiple genes simultaneously in the rice
genome
CRISPR/Cas systems: The link between
functional genes and genetic
Improvement- RICE AS REFERENCE

13. • Grain yield is determined mainly by three
components: panicle number, grain number per
panicle, and grain weight
1.Genome editing to increase rice yield
1.1 Knockout of yield-reducing genes to increase rice yield
that reduce yield in elite rice cultivars via gene-editing technology is a
promising approach to the creation of ideal rice cultivars. 4 yield-reducing
functional genes:
• GRAIN NUMBER 1a (Gn1a)
• DENSE AND ERECT
• PANICLE1 (DEP1)
• GRAIN SIZE 3 (GS3)
• IDEAL PLANT ARCHITECTURE1 (IPA1),

14. 1.2Manipulation of regulators of cytokinin homeostasis and
signal response to increase rice yield
• New rice germplasm with increased salinity tolerance
without loss of grain yield was obtained by CRISPR/Cas9-
targeted mutagenesis of the OsRR22 gene, which is
involved in both cytokinin signal transduction and
metabolism
• CRISPR-edited variants at the 30-end of OsLOGL5, which
encodes a cytokinin-activation enzyme, increased grain
yield under various field environments
• Cytokinin oxidase/dehydrogenase (CKX) is the main
enzyme that inactivates cytokinin. Disruption of OsCKX11
using the CRISPR/Cas9 system increased cytokinin content
and grain yield compared with wild-type rice

15. Strategies for improving rice yield using gene-editing
technology
OsCKX2/Gnla is the common target gene of two strategies (functional genes for
decreasing yield and cytokinin-homeostasis and signal-response regulators)

16. • Simultaneous mutation of rice genes encoding the abscisic acid
(ABA) receptors PYRABACTIN RESISTANCE 1-LIKE 1
(PYL1), PYL4, and PYL6 yielded plants with robust growth and
increased grain yield
• CRISPR/Cas9 induced modification of PYL9, which encodes one
of the rice ABA receptors, increased drought tolerance and grain
yield
• New rice cultivars with both high yield and high cold tolerance
were obtained by simultaneously editing two yield-related negative
genes (OsPIN5b and GS3) and one cold-tolerance gene
(OsMYB30) using the CRISPR/Cas9 system
• Rice PARAQUAT TOLERANCE 3 (OsPQT3) knockout mutants
generated using CRISPR/Cas9 technology conferred higher yield
and increased resistance to oxidative and salt stress
1.3 Editing of plant-growth and environmental-
response regulators to increase rice yield

17. 1.4 Disruption of microRNA regulators to increase
rice yield
• Knockout of MIR396e and MIR396f via CRISPR/Cas9
increased both grain size and panicle branching,
resulting in increased grain yield
• Mutation in OsGRF4 introduced by CRISPR/Cas9
perturbed the miR396-directed regulation of OsGRF4
and generated plants with larger grain size and increased
grain yield similar to those reported previously
• Knockout of UCLACYANIN 8 (OsUCL8), the
downstream target of miR408, led to increased grain
yield

18. • Rice grain quality is evaluated mainly based on four
quality standards: processing quality, appearance
quality, eating and cooking quality (ECQ), and
nutritional quality
2. Genome editing to improve rice grain
quality
Improvement of rice grain appearance quality, eating
and cooking quality and grain nutritional quality
• Knockout of GS3 and GL3.1 rapidly improved grain size
• GS3 and Gn1a, which controls grain number, were successfully edited
in four rice cultivars.
• Both gs3 and gs3gn1a mutants exhibited greater grain length and 1000-
grain weight than their wild-type counterparts, and the gs3gn1a double
mutants had more grains per panicle than the gs3 mutants

19. • Loss-of-function mutants generated using the CRISPR/Cas9
system to target the Wx coding region in multiple rice
cultivars all showed decreased AC (Amylose Content) and
produced glutinous rice
• Knockout of the phospholipase D gene (OsPLDa1) using
the CRISPR/Cas9 system produced rice grains with reduced
phytic acid content compared with their wild-type
counterparts
• Knockout of the OsNramp5 gene using the CRISPR/Cas9
system produced promising rice cultivars with extremely
low cadmium accumulation in grains, without
compromising yield

20. Genome editing to improve rice grain quality

21. 3. Genome editing to improve rice resistance
3.1 Herbicide resistance
• Introduction of point mutations into the rice ALS gene using
CRISPR/Cas9-mediated homologous recombination also conferred
herbicide tolerance on rice plants
• The 548th and 627th amino acids of the rice ALS gene were
• edited to yield novel rice genotypes with bispyribac-sodium
resistance
• Other amino acid substitutions in ACCase, such as W2125C and
P1927F, were also obtained by CRISPR-based saturated targeted
endogenous mutagenesis editors, which conferred haloxyfop
resistance in rice
• The T102I and P106S aminoacid mutations in EPSPS and the
M268T mutation in TubA2 have been reported to confer rice
resistance to glyphosate and trifluralin, respectively

22. 3.2 Disease and insect-pest resistance
• Li et al., 2020 used the CRISPR/Cas9 system to edit the Xa13
promoter and obtained transgene-free bacterial-blight-resistant rice
• CRISPR/Cas9-directed mutagenesis in the
Xa13/Os8N3/OsSWEET11 coding region can also confer resistance
to Xanthomonas oryzae pv. Oryzae (Xoo)
• CRISPR/Cas9 editing in an ethylene responsive factor, OsERF922,
which is a negative regulator of blast resistance, showed increased
resistance to Magnaporthe oryzae without alteration to agronomic
traits
• Lu et al., 2018 used CRISPR/Cas9 technology to knock out the
cytochrome P450 gene CYP71A1, encoding tryptamine 5-
hydroxylase, which catalyzes the conversion of tryptamine to
serotonin, thereby leading to rice resistance to insect pests via
suppression of serotonin biosynthesis

23. Genome editing to improve rice resistance

24. Exploitation and utilization of heterosis
• Shen et al., 2019 rapidly created marker-free photoperiod
/thermosensitive genic male sterile (P/ TGMS) rice materials by
editing the male fertility gene PTGMS2-1 in two widely compatible
rice cultivars
• Japonica photosensitive genic male-sterile rice lines were developed
by targeted editing of the Carbon Starved Anther gene in japonica
rice using CRISPR/ Cas9
• Thermosensitive male sterile lines were also created by mutagenesis
of the TMS5 gene by the CRISPR/Cas9 system
• Li et al., 2019 developed disease-resistant thermosensitive male-
sterile rice by simultaneous genome engineering of the TMS5, Pi21,
and Xa13 genes
• Knockout of the SaF and SaM alleles by CRISPR/Cas9 produced
hybrid-compatible lines that overcame Sa-mediated hybrid male
sterility in rice

25. Applications of CRISPR/Cas system on gene
function study