Multi Target Gene Editing using CRISPR Technology for Crop Improvement

Plant Biotechnology Centre , DBSKKV, Dapoli
A
Presentation
On
Multi Target Gene Editing using CRISPR Technology for
Crop Improvement
Presented by
Gajare Tushar .P.
Reg.No. 0030
Sr.Msc. Agricultural Biotechnology
Plant Biotechnology Centre
College of Agriculture, Dapoli

CRISPR-Cas
• Introduction
• CRISPR
• Cas
• How it works?
• CRISPR-Cas: A tool for Genetic Engineering
• History
• Applications
• Applications in Crop Improvement
• Case studies
• Advantages
• Disadvantages
• Safety issues
• Future apsects
• Conclusion
• References

Introduction
• Genome editing :
Genome editing or gene editing, is a group of
technologies that give scientists the ability to change an
organism's DNA.
• These technologies allow genetic material to be added,
removed, or altered at particular locations in the
genome.
• Several approaches to genome editing have been
developed. Eg: CRISPR, TALENs, ZFNs

Introduction
• What is CRISPR-Cas?
• CRISPR-Cas(9) is a unique technology that enables us
to edit parts of the genome by removing, adding or
altering sections of the DNA sequences.
• It is currently the simplest, most versatile and precise
method of genetic manipulation and is therefore
causing a buzz in the science world.

CRISPR
• Clustered Regularly Interspaced Short Palindromic
Repeats.
• It is a family of DNA sequences found within the genomes
of prokaryotic organisms such as bacteria and archaea.
• Derived from DNA fragments from viruses that have
previously infected the prokaryote and are used to detect
and destroy DNA from similar viruses during subsequent
infections.
• Plays a key role in the antiviral defence system of
prokaryotes.

Cas9
• CRISPR-associated protein 9.
• An enzyme that uses CRISPR sequences as a guide to recognize
and cleave specific strands of DNA that are complementary to
the CRISPR sequence.
• Cas9 enzymes together with CRISPR sequences form the basis of
a technology known as CRISPR/Cas9 that can be used to edit
genes within organisms.
Cas9 protein molecule

How it works?
• In this system, the endonuclease of Cas9 is directed to
DNA targets by a guideRNA .
• The ribonucleoprotein complex of Cas9 and gRNA
recognizes the DNA sequence that is complementary to
the 5’-end of the guideRNA.
• The DNA sequence is then cleaved.
• Thus disintegrating the viral DNA.

CRISPR-Cas(9): As a Tool
• Cas9 (acts as a pair of ‘molecular scissors’ ) cuts the two strands of DNA at a
specific location in the genome.
• The bits of DNA are then be added or removed.
• gRNA consists of a small piece of predesigned RNA sequence (about 20 bases
long) located within a longer RNA scaffold.
• Pre-designed sequence ‘guides’ Cas9 to the right part of the genome.
• This makes sure that the Cas9 enzyme cuts at the right point in the genome.
• The guide RNA is designed to find and bind to a specific sequence in the DNA.
• gRNA has bases complementary to the target DNA molecule.
• the guide RNA will only bind to the target sequence and no other regions of
the genome.
• The scaffold part binds to DNA .
• The Cas9 follows the guide RNA to the same location in the DNA sequence
and makes a cut across both strands of the DNA.
• At this stage the cell recognises that the DNA is damaged and tries to repair it.
• DNA repair machinery is used to introduce changes to one or more genes the
genome by adding , deleting or replacing the genes.

Cas9
(acts as a pair of ‘molecular
scissors’ )
cuts the two strands of DNA at a
specific location in the genome.
The bits of DNA are then be added
or removed
gRNA
It ‘guides’ Cas9 to the right part of
the genome.
This makes sure that the Cas9
enzyme cuts at the right point in
the genome.
The scaffold part binds to DNA .
The Cas9 follows the guide RNA to
the same location in the DNA
sequence and makes a cut across
both strands of the DNA.
cell recognises that the DNA is
damaged and tries to repair it. DNA repair machinery is used
Genome is edited by adding ,
deleting or replacing the genes.

History
• The discovery of clustered DNA repeats occurred independently in three
parts of the world.
1987
• Yoshizumi Ishino et al.
• Accidentally cloned part of a CRISPR together with the iap gene, the target of interest.
• They studied the relation of "iap“ to the bacterium E. coli.
1993
• Mycobacterium tuberculosis
• In Neterlands two research articles
• Diversity of cluster of interrupted direct repeats in different strains
• spoligotyping
1993
• Francisco Mojica
• Observed and studied the function of repeats in Haloferax and Haloarcula species

2000
•Mojica
•Survey of scientific literature
2001
•Mojica and Ruud Jansen
•Proposed the acronym CRISPR
2002
•Tang, et al.
•showed evidence that CRISPR repeat regions from the genome of Archaeoglobus
fulgidus were transcribed into long RNA molecules that were subsequently
processed into unit-length small RNAs, plus some longer forms of 2,3, or more
spacer-repeat units.

2013
•Cong et al
•First event of CRISPR/Cas 9 in Eukaryotes
•Mao et al
•designed two sgRNAs for the photosynthesis in Arabidopsis thaliana.
2014
•Wang et al
•Cas 9 used for functional screening
2015
•Ousterout et al
•Multiplex CRISPR/Cas 9

Applications
• Biomedical research
• Treating diseases and genetic disorders
• Crop Improvement

Applications in Crop Improvement
• Gene knockouts:
• Stress resistance:
• Inducing crop diversity:
• In nitrogen fixation:
• To reduce apple acidity:
• Inducing polyploidy:
• For suppressing viral infection:
• Muti target gene editing/ Multiplex Genome
editing :

• Gene knockouts:
To eliminate undesirable genes
• Stress resistance:
Multiplexing ability to impart biotic and abiotic stress tolerance.
Eg: virus-induced gene silencing (VIGS) in tomato.
• Inducing crop diversity:
• To create a high degree of genetic variability at a precise locus in the
genome of the crop plants.
• It is a potential tool for multiplexed reverse and forward genetic study.
• Genome editing allows precise and predictable modifications directly in elite
cultivars or accessions, saving the time consuming backcrossing procedure in
conventional breeding schemes.

• In nitrogen fixation:
• To reduce apple acidity:
• Inducing polyploidy:
• For the creation and use of novel allelic variants for
breeding in crops.
• Production of haploids.
• Generating Polyploidy
• E.g: Potato and wheat.
• For suppressing viral infection:
Highly conserved sequences of Gemini viruses have been
targeted using CRISPR/Cas9 to good effect (Ali et al., 2016).

Muti target gene editing/ Multiplex
Genome editing : WHY ?
• In plants, cellular processes are fine-tuned by
several genes.
• Sometimes, mutating a single gene may not
confer a desired phenotype because of the
compensation effect produced by other genes
in same gene family.
• Hence, an upgraded editing system with
improved efficiency is needed for multiplex
gene editing in plants.

Case Studies
1
Targeted Mutagenesis, Precise Gene Editing,
and Site-Specific Gene Insertion in Maize Using
Cas9 and Guide RNA.
Svitashev, S., Young, J. K., Schwartz, C., Gao, H.,
Falco, S. C., & Cigan, A. M. (2015).

• Crop : Zea mays
• Target genes : Liguleless1 (LIG1) gene, male fertility genes
(Ms26 and Ms45), and acetolactate synthase (ALS) genes
(ALS1 and ALS2)
• Cas9 promoter: ZmUbi
• Codon optimazition of Cas9 : Maize
• sgRNA promoter : ZmU6
• Transformation method :
Biolistic transformation
• Multiplex strategy : Co-delivery
• Mutant efficiancy : 77–100%
• Type of mutant : biallelic,heterozygous

Method
• Targeted mutagenesis, editing of endogenous maize
genes, and site-specific insertion of a trait gene using
Cas9-guide RNA technology is reported in Zea mays.
• DNA vectors expressing maize codon-optimized
Streptococcus pyogenes Cas9 endonuclease and single
guide RNAs were co-introduced with or without DNA
repair templates into maize immature embryos by
biolistic transformation targeting five different
genomic regions: upstream of the liguleless-1 gene
(LIG), male fertility genes (MS26 and MS45) and 49
acetolactate synthase genes (ALS1 and ALS2).

Observations
• Mutations were subsequently identified at all
sites targeted
• Plants containing biallelic multiplex mutations
at LIG, MS26 and MS45 were recovered.

Results
• Biolistic delivery of guide RNAs (as RNA molecules) directly
into immature embryo cells containing pre-integrated Cas9
also resulted in targeted mutations.
• Editing the ALS2 gene using either single-stranded
oligonucleotides or double-stranded DNA vectors as repair
templates yielded chlorsulfuron resistant plants.
• Double-strand breaks generated by RNA guided Cas9
endonuclease also stimulated insertion of a trait gene at a
site near liguleless-1 by homology-directed repair.
• Progeny demonstrated expected Mendelian segregation
of mutations, edits, and targeted gene insertions.

Conclusion
• The examples reported in this study
demonstrate the utility of Cas9-guide RNA
technology as a plant genome editing tool to
enhance plant breeding and crop research
needed to meet growing agriculture demands
of the future

Case Study 2
Lycopene Is Enriched in Tomato Fruit
by CRISPR/Cas9-Mediated Multiplex
Genome Editing
Xindi Li
1
, Yanning Wang
1
, Sha Chen
2
, Huiqin Tian
1
, Daqi Fu
1
, Benzhong Zhu
1
,
Yunbo Luo
1
and Hongliang Zhu
1
* (2018)

• Crop : Tomato (Lycopersicon esculentum)
• Target genes : 5
1. SGR1
2. lycopene ε-cyclase (LCY-E)
3. beta-lycopene cyclase (Blc)
4. lycopene β-cyclase 1(LCY-B1)
5. LCY-B2
• Target sites : 6
• Promoters: pAtU3d, pAtU3b, pAtU6-1,pAtU6-29
• Transformation method : Agrobacterium tumefaciens-mediated
transformation
• Vector : pYLCRISPR/Cas9-Lycopene vector (binary plasmid )
• Multiplex strategy : bidirectional strategy
• Type of mutant : biallelic,homozygous

• Numerous studies have been focusing on breeding tomato plants with
enhanced lycopene accumulation, considering its positive effects of fruits
on the visual and functional properties.
• In this study, a bidirectional strategy: promoting the biosynthesis of
lycopene, while inhibiting the conversion from lycopene to β-and α-
carotene was used.
• The accumulation of lycopene was promoted by knocking down some
genes associated with the carotenoid metabolic pathway.
• Finally, five genes were selected to be edited in genome by CRISPR/Cas9
system using Agrobacterium tumefaciens-mediated transformation.
• Findings indicated that CRISPR/Cas9 is a site-specific genome editing
technology that allows highly efficient target mutagenesis in multiple
genes of interest.
• The lycopene content in tomato fruit subjected to genome editing was
successfully increased to about 5.1-fold.
• The homozygous mutations were stably transmitted to subsequent
generations.

FIGURE : Selection of target genes and
designing of CRISPR/Cas9 binary expression
cassette.
(A) A map of the target genes in the
carotenoid metabolic pathway. The green
boxes represent the key substances in the
metabolic pathway. The red and orange
boxes show the two substances, lycopene
and β-carotene, respectively. A solid arrow
indicates a direct effect, and a dashed arrow
indicates an indirect effect. The selected
target genes are represented by purple
boxes, and the red asterisks represent the
sites at which the target genes act on the
pathway. G3P, glyceraldehyde 3-phosphate;
DXS, 1-deoxy-D-xylulose 5-phosphate
synthase; GGPPS, geranylgeranyl
pyrophosphate synthase; PDS, phytoene
desaturase; ZISO, z-carotene isomerase

(B) Five target genes were selected
according to the synthesis and
metabolism pathways of lycopene,
and six target sites were designed.
The target sequences are marked in
red, and small rectangle frames
indicate the PAM. Straight lines and
boxes are the introns and exons of
the target genes, respectively.

(C) Structures of the pYLCRISPR/Cas9-Lycopene binary
vectors. HPT(−H) encodes hygromycin B phosphotransferase.
The six targets designed are represented by solid boxes in
different colors, and the promoters used for each target are
shown.

Methodology
Plant Material
(Leaves )
Selection of sgRNA
Target Sequence
pYLCRISPR/Cas9-
Lycopene Vector
Construction
Plant Transformation
(Agrobacterium-
mediated )
DNA Extraction and
Mutation Detection
Cas9 and Off-Target
Analysis
Carotenoid
Extraction and RT-
HPLC Analysis
Carotenoid
Extraction and HPLC-
MS Analysis
Transmission
Electron Microscope
Analysis

Result

Conclusion
• The results suggest that CRISPR/Cas9 system
can be used for signiﬁcantly improving
lycopene content in tomato fruit with
advantages such as high efﬁciency, rare off-
target mutations, and stable heredity.

Advantages
• Faster
• Cheaper
• More accurate
• Efficient
• Specific
• Less occurance of unwanted effects than other
existing genome editing methods.
• Ability of multiple targeting simultaneously.

DisAdvantages
• Potential threat to Environment.
• Risk of Permanent Modification.
• Concerns have been raised that off-target effects (editing
of genes besides the ones intended) may obscure the
results of a CRISRP gene editing experiment (the
observed phenotypic change may not be due to
modifying the target gene, but some other gene).
• Modifications to CRISRP have been made to minimize the
possibility of off-target effects.
• In addition, orthogonal CRISPR experiments are
recommended to confirm the results of agene editing
experiment.

Issues
• Safety
• Ethical
• Social
• Political
• Acceptance

Future apsects
• Gene editing
• Transcription
• Chromosome structure Determination

Conclusion
• CRISPR-Cas(9) enables us to edit genome by
targeting multiple genes in a single approach with –
• Great ease
• High specificity
• Higher speed and accuracy.
• Minimal or no off-targets side effects.
• It is currently the simplest, most versatile and
precise method of genetic manipulation and is
therefore causing a buzz in the science world.

References
• Devashish Rath , Amlinger. L. , Rath. A. , Lundgren.M. (2015)The CRISPR-Cas immune system: Biology,
mechanisms and applications. Biochime(17):119-128.
• Tsai S..Q, Zheng Z., Nguyen N.T., Liebers M., Topkar V.V., Thapar V. (2015) GUIDE-seq enables genome-wide
profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 33 (2):187–197.
• Svitashev, S., Young, J. K., Schwartz, C., Gao, H., Falco, S. C., & Cigan, A. M. (2015). Targeted Mutagenesis,
Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA. Plant Physiology,
169(2), 931–945. doi:10.1104/pp.15.00793
• Lowder, L. G., Zhang, D., Baltes, N. J., Paul, J. W., Tang, X., Zheng, X., … Qi, Y. (2015). A CRISPR/Cas9 Toolbox for
Multiplexed Plant Genome Editing and Transcriptional Regulation. Plant Physiology, 169(2), 971–985.
• Yang, Q., Zhang, C., Chan, M., Zhao, D., Chen, J., Wang, Q., … Liu, Q. (2016). Biofortification of rice with the
essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance. Journal
of Experimental Botany, 67(14), 4285–4296. doi:10.1093/jxb/erw209
• Ali Z., Ali S., Tashkandi M., Zaidi S.S., Mahfouz M.M. (2016) CRISPR/Cas9-mediated immunity to geminiviruses:
differential interference and evasion. Sci Rep. 6.
• Doench J.G., Fusi N., Sullender M., Hegde M., Vaimberg E.W., Donovan K.F., SmithI.,Tothova Z., Wilen C. (2016)
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol.
34:184–191.
• Gayatonde, V., &Vennela, P. R. (2017). CRISPR-Cas; A potential technique for crop improvement. Biotech
Express. 4(34):34-38.
• Antre S. H., Mandlik R. R., Guru P. N.(2017) CRISPR/Cas9 : A Game Changer Tool in Crop Improvement. Current
Advances and Challenges in Agricultural Biotechnology(pp.). PRES’s College of Agricultural Biotechnology, Loni,
Ahmednagar.

• Georges, F., & Ray, H. (2017). Genome Editing of Crops: A Renewed Opportunity
for Food Security. GM crops & food, 0.
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Significance, opportunities and recent advances. Plant Physiology and
Biochemistry.
• Li, X., Wang, Y., Chen, S., Tian, H., Fu, D., Zhu, B., … Zhu, H. (2018). Lycopene Is
Enriched in Tomato Fruit by CRISPR/Cas9-Mediated Multiplex Genome Editing.
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• Leong, K.-Y. B., Chan, Y.-H., Abdullah, W. M. A. N. W., Lim, S.-H. E., & Lai, K.-S.
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Multi Target Gene Editing using CRISPR Technology for Crop Improvement

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