Developing fnCas9 vector for genome editing in rice through tissue culture

Dissertation Presentation on topic
Developing fnCas9 Vector for Genome Editing in Rice through
Tissue Culture
PRANAY UPADHYAY
M.Sc. Biotechnology 4th Semester
Class Roll no – 01
Registration no – 2020103704
University Roll no- 2120140150009
Anugrah Narayan College Patna, (Bihar)
Dr.Hasthi Ram
(Staff Scientist III)
NIPGR, New Delhi

OUTLINE
 Problem Statement
 Introduction
 Need for the study
 Objectives
 Hypothesis
 Assumptions
 Review of Literature
 Methodology
Interpretation of Data
 Discussion
 Summary
 Conclusion

Problem Statement
The study was to develop an efficient and
precise fnCas9 vector delivery system that can
be used for genome editing in rice through
tissue culture, to generate improved rice
varieties with desirable traits that can overcome
biotic and abiotic stresses, ultimately
contributing to global food security.

A Quick Introductory Review
 In recent years, genome editing has emerged as a powerful tool for precision
breeding of crops, and the Cas9 enzyme is at the forefront of this technology.
 fnCas9 and spCas9 are two of the most widely used Cas9 variants, and their
effectiveness in rice tissue culture is of particular interest to the agricultural
industry.
 In this presentation, we will explore the principles of genome editing with
fnCas9 and introduction of SG-2 gene with spCas9 in MTU1010 variety rice
through tissue culture via Agrobacterium mediated gene transfer method in rice
calli through callus culture using pRGEB32 comprised of spCas9.
 We will also discuss the advantages and limitations of each variant, and the
current state of research in this exciting field. So, let's dive in and discover the
exciting possibilities of fnCas9 and spCas9 in rice tissue culture.

Need For The Study
Molecular Biology
 The study of genome editing through the fnCas9 vector is important
because it provides a powerful tool for scientists to make precise
modifications to DNA sequences in living cells.
 fnCas9 is a type of Cas9 protein that has been engineered to be smaller
and more precise than the original Cas9 protein. This makes it an attractive
tool for genome editing because it can be delivered more easily to cells and
has a lower risk of off-target effects.
 By studying genome editing through the fnCas9 vector, researchers can
learn more about how this technology works, how to optimize its efficiency
and accuracy, and how to minimize off-target effects. This knowledge can
help to accelerate the development of new therapies for genetic diseases, as
well as new approaches for improving crops and livestock.

Plant Tissue Culture
 Improving crop yield: The genetic modification of rice through the
agrobacterium-mediated gene transfer method can improve crop yield by
introducing genes that enhance the plant's ability to resist pests and diseases,
tolerate abiotic stresses such as drought and salinity, and increase nutrient
uptake.
 Developing new rice varieties: The Agrobacterium-mediated gene transfer
method can be used to introduce desirable traits into rice, such as disease
resistance, herbicide tolerance, and improved quality characteristics.
 Basic research: Studying the plant tissue culture of rice through the
Agrobacterium-mediated gene transfer method can provide insights into the
molecular and cellular mechanisms underlying plant growth, development, and
response to environmental stress.
 Conservation of rare or endangered rice species: The agrobacterium-
mediated gene transfer method can be used to conserve rare or endangered rice
species by preserving their genetic material in tissue culture and introducing it
into other plants.

Core Objectives
 To design and construct a functional Cas9 nuclease
(fnCas9) vector for efficient and precise genome editing in
rice plants.
 To optimize the tissue culture conditions for rice callus
induction, regeneration, and transformation through
Agrobacterium-mediated gene transfer method.
 To evaluate the efficiency and specificity of the spCas9
system in generating targeted mutations or gene knockouts in
rice plants.
 To assess the stability and heritability of the edited traits in
the subsequent generations of the transformed rice plants. Plant tissue culture(Callus culture) in rice

Hypothesis
 The pRGEB32 vector with spCas9, when
combined with optimized tissue culture
conditions and Agrobacterium-mediated
gene transfer method, will enable efficient
and precise genome editing in rice plants.
 The edited traits will be stable and
heritable in the subsequent generations of
the transformed plants, and the fnCas9
system will prove to be a valuable tool for
improving the agronomic traits and
productivity of rice plants. Genome editing strategies using CRISPR/Cas9 showing
HITI approach and NHEJ repair

Assumptions
 The tissue culture conditions
and Agrobacterium-mediated
gene transfer method will be
optimized to ensure efficient
transformation and regeneration
of rice plants.
 The edited traits will be stably
inherited.
 The fnCas9 vector will function
as intended.
CRISPR system depicting the SpCas9 system,
which detects NGG PAM sequences and
produces blunt-ended DSBs, is the most widely
utilised CRISPR system.

Review of Literature
This work focuses on the development of the
fnCas9 vector for genome editing alongwith
successful introduction of SG-2 gene in rice
through callus culture via Agrobacterium-
mediated gene transfer. The development work of
fnCas9 vector as a more specific alternative to
spCas9, with reduced off-target effects and
increased on-target activity, of mine, was in still
in progress. The use of tissue culture in
conjunction with Agrobacterium-mediated gene
transfer allows for the production of large
numbers of genetically identical plants from a
single transformed cell, making it a powerful tool
for crop improvement.
The No. of publications explaining the
application of the TALEN, ZFN, and
CRISPR/Cas9 technologies. During the
past several years.

ZFN TALEN
CRISPR/Cas
This approach is a promising method for
improving rice varieties and could lead to
more efficient genome editing.
 Genome editing is a powerful tool for
crop improvement.
 ZFN, TALEN, and CRISPR/Cas9 are the
three main actors in genome editing
technologies.
 CRISPR-Cas9 is a widely used genome
editing system.
 Rice is a major staple crop, making it an
important target for genome editing.

Methodology
 Design and construction of the fnCas9 vector.
 Optimization of tissue culture conditions for rice callus
induction, regeneration, and transformation through
Agrobacterium-mediated gene transfer method.
 Competent Cells Efficiency Check by transforming pRGEB32
into E.coli.
 Transformation of 3xHA fnCas9 and pRGEB32 into E.coli.
 Cloning of gRNA scaffold into pJET1.2 Blunt End cloning
vector.

PCR Amplification of 3XHA fnCas9, pRGEB32 & pJET+gRNA
Scaffold.
 SANGER SEQUENCING
 SINGLE, DOUBLE and SEQUENTIAL Digestion of Vectors
using different enzymes.
 Side Directed Mutagenesis (SDM) of PCR amplified product
from Digestion of fnCas9 plasmid with BamHI and SacI
 Evaluation of the efficiency and specificity of the spCas9 system
in generating targeted mutations or gene knockouts in rice plants.

Interpretation of Data
The designed SG-2 gene with desired characteristics was ligated into pRGEB32 plasmid,
which was serving as a host plasmid, and later introduced into the rice genome via the
Agrobacterium-mediated gene transfer method through plant tissue culture.
Plant Tissue Culture

Surface sterilization and Inoculation
of MTU1010, indica variety of rice
seeds was done.
Callus induction in rice seeds was
observed after 7 days of inoculation

Subculture of Calli after 15 days
of inoculation.
Rice calli infected with
Agrobacterium having SG2 gene
and kept on CC plates for 48 hr.

After Co-Cultivation calli were shifted to
section plates after washing.
After completion of 15 days in selection 1
the calli were shifted on selection 2.

After completion of 15 days in selection 2 the calli were
shifted on third selection plates.

Molecular Work
pRGEB32 having spCas9 3xHAFnCas9

Competent Cells Efficiency Check by transforming pRGEB32
 pRGEB32 containing spCas9 was trnsformed
into competent cells successfully and effectively.
 To verify successful transformation, newly
produced competent cells and a selective
medium containing kanamycin were used.
 To increase the effectiveness of the even
further, the experimental setup was perfected
along with the cautious management of the
competent cells and DNA by placing them in
-70℃.

Transformation of 3XHA fnCas9 and pRGEB32
3XHAfnCas9 CFU’s pRGEB32 CFU’s
 The plasmids 3XHAfnCas9 and pRGEB32, were both effectively transformed into
ampicillin and kanamycin plates, respectively.
 They had a sizable colony population, as suggested by the literary evidence and
were additionally kept and segregated for later use.

PCR amplification of fnCAS9 from
3xHA fnCas9 vector
Initial Denaturation 980C 1 min
Denaturation 980C 10 sec
Annealing 600C 15 sec
Elongation 680C 6 min
Final Elongation 680C 10min
Infinite Holding 120C ∞
 Expected Band Size was
5.8 kb.
 False band was observed
at around +3 kb.

Elongation 680C 40 sec
Final Elongation 680C 10 min
 Expected Band Size was 134 bp.
PCR amplification of fnCAS9gRNA scaffold
from 3xHA fnCAS9 vector

Cloning of gRNA scaffold into pJET1.2 Blunt End:
Before transformation After transformation
 Successful cloning of gRNA scaffold sequence into the pJET1.2 Blunt end vector
was done with good efficiency, representing an important step in the development of
the CRISPR-Cas9 system for genome editing.
 The presence of the gRNA scaffold sequence in the pJET1.2 Blunt end vector was
later confirmed by restriction digestion and Sanger sequencing.

SANGER SEQUENCING
 pJET1.2 blunt+gRNA scaff.dna alignment from 1 to 3116. Found positive
colony having gRNA scaffold ligated into pJET1.2 blunt.
3xHA fnCas9 gRNA scaffold region

 Expected Band Size was 134bp.
Double Digestion of pJET+gRNA scaffold by Bsa1 and Sbf1
Plasmid
1
Plasmid
2

Double Digestion of 3X HAFncas9 by BamH1 and Sac1
Expected band size was 5.8 kb & 4.2 kb. Band of 5.8kb was cut and eluted from gel.

Elongation 680C 2 min 20 sec
Final Elongation 680C 10 min
PCR amplification of fragment from Digestion of fnCas9 plasmid with
BamHI and SacI (SDM)
Expected Band Size was 2190 bp.

Sequential Digestion of pRGEB32 by BstB1 and Xba1
Expected band size was 11.550 kb &
4.338 kb band was to be removed.

Double Digestion of PRGEB32 by Bsa1 and Sbf1
to get gRNA Scaffold
 Expected band size was 15.75 kb & 148 bp.
148 bp was to be removed but we didn’t get any
band.

3xHA fnCAS9 vector
 Expected band size was 5.8 kb.
 We couldn’t succeed to amplify
the desired sequence.

PCR amplification of fnCAS9 from 3xHA fnCAS9 vector

3xHA fnCAS9 vector

Discussion
 fnCas9: a promising genome editing tool for plants, including
rice- Agrobacterium-mediated gene transfer: widely used method
for introducing foreign DNA into plants.
 Combining FnCas9 with agrobacterium-mediated gene transfer
could improve efficiency and precision of genome editing in rice.
 Tissue culture: essential for regenerating transformed plants
from single cells for screening of desired genetic changes.
 Aims to develop an efficient and precise genome editing tool
for rice.
 Significance: potential contribution to crop improvement and
global food security

Summary
The dissertation topic focuses on the development of a vector system
based on the fnCas9 nuclease for efficient genome editing in rice.
The another vector system comprised of spCas9 system was used in
callus culture to deliver SG-2 gene in rice cells via Agrobacterium-
mediated gene transfer method. The study aims to optimize the
protocol for rice tissue culture and transformation, evaluate the
efficacy of spCas9-mediated genome editing in rice, and establish a
stable genome-edited rice line alongwith the development of a vector
system based on the fnCas9. The results of this research could
contribute to the development of improved methods for genome
editing in rice, a major staple crop that feeds much of the world's
population.

Conclusion
 Developing a fnCas9 vector for genome editing will perish new posibilities
for scientists to generate new varieties in rice instead of using spCas9 which is
already patented. After this we can use tissue culture which has great potential
for improving crop yields and resilience.
 Further research is needed to optimize the delivery of the spCas9 vector into
rice cells through tissue culture.
 In conclusion, the development of a fnCas9 vector for genome editing & use
of pRGEB32/spCas9 for delivering SG-2 gene in rice through callus culture is
an effective approach for generating mutations in rice. This technique can be
used to improve crop yield, increase disease resistance, and enhance nutritional
quality in rice.

References
F. Adikusuma et al., (2017) Development and Applications of CRISPR/Cas9 Genome Editing
Technology.
Kogawa et al., Nishikawa et al., Saeki et al and Yoda et al., (2023)
Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read
sequencing.
Mohanty et al., (2021) Sequencing of diverse genomes from the NHLBI TOPMed Program.
Maeder et al., r and Gerbach et al., (2016) Genome-editing Technologies for Gene and Cell
Therapy.
Jinek et al., (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial
immunity.
Cong et al. and Mali et al., (2013) Development and Applications of CRISPR-Cas9 for Genome
Engineering.
Barrangou et al., and Doudna et al., (2016) Applications of CRISPR technologies in research and
beyond.
Zhang et al., (2016) Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated
homologous recombination of acetolactate synthase.
Zhang et al., (2019) Plant Prime Editors Enable Precise Gene Editing in Rice Cells.
Wang et al., (2020) Quantitative regulation of Waxy expression by CRISPR/Cas9‐based promoter
and 5'UTR‐intron editing improves grain quality in rice.
Li et al., (2017) Improved Base Editor for Efficiently Inducing Genetic Variations in Rice with
CRISPR/Cas9- Guided Hyperactive hAID Mutant.

 Zhou et al., (2019) Cytosine, but not adenine, base editors induce genome-wide off-target
mutations in rice.
Li et al., Liu et al., Spalding et al., Weeks et al., Yang et al., Zhang et al., Wei et al., Zhang
et al., Gou et al., Feng et al., Mao et al., and Yang et al., (2017).
Shan et al., (2015) Creation of fragrant rice by targeted knockout of the OsBADH2 gene
using TALEN technology.
Tang et al., (2017) Genome-wide Targeted Mutagenesis in Rice Using the CRISPR/Cas9
System.
Komor et al., (2017) A CRISPR/Cas9 toolkit for efficient targeted base editing to induce
genetic variations in rice.
Wang et al., (2017) Expanding the Scope of CRISPR/Cpf1-Mediated Genome Editing in
Rice.
Xu et al. (2016) Genome Editing in Rice: Recent Advances, Challenges, and Future
Implications.
Thakur et al., kaur et al., kapoor et al., and salvi et al., (2022). Efficient Genetic
Transformation of Rice for CRISPR/Cas9 Mediated Genome-Editing and Stable
Overexpression Studies.
 Zhou et al., (2020) A platinum standard pan-genome resource that represents the
population structure of Asian rice.

PRANAY UPADHYAY
upadhyaypranay15@gmail.com

Developing fnCas9 vector for genome editing in rice through tissue culture

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