Targeted genome editing by use of artificial nucleases has the plausible potential to speed basic research as well as plant breeding by providing the means to modify genomes quickly in a specific and predictable manner but advanced CRISPR-Cas9 based technologies first confirmed in mammalian cell systems are quickly being fitted for use in plants. These new technologies increase CRISPR-Cas9’s utility and effectiveness by diversifying cellular capabilities through expression construct system evolution and enzyme orthogonality, as well as enhanced efficiency through delivery and expression mechanisms. RNA-guided genome editing using Streptococcus pyogenes CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) has renewed the concept of genome editing in plants. CRISPR-associated surveillance complexes are easily programmable molecular sleds that can target any sequence of choice. These complexes offer new opportunities for implementation in biotechnology. Recent studies have used CRISPR/Cas9 to engineer virus resistance in plants, either by directly targeting and cleaving the viral genome, or by modifying the host plant genome to introduce viral immunity. The CRISPR/Cas9 platform could also be used for targeted mutagenesis to identify host factors that control plant resistance and susceptibility to viral infection. Thus, CRISPR/Cas9 technology offers a promising approach for under- standing and engineering resistance to single and multiple viral infections in plants.

1. Engineering plant immunity using CRISPR/Cas9 to
generate virus resistance
CRISPR-CAS9
Presented By:
Sheikh Mansoor Shafi
Ph.D Biochemistry
Reg. No: J-16-D-09-BS

2. •WHAT IS CRISPR CAS9?
• KEY COMPONENTS OF CRISPER
• NATURAL CRISPR SYSTEM BACTERIA
•GENERAL PROTOCOL
•DELIVERY OF CRISPR REAGENTS TO PLANT CELLS AND TISSUES
•The CRISPR APPROACHES TO MAKE VIRUS RESISTANT PLANTS
•EXAMPLES OF CROPS MODIFIED WITH CRISPR TECHNOLOGY
•CASE STUDIES
•APPLICATIONS
•FUTURE PROSPECTS
•CONCLUSION
CONTENTS

3. The field of site-specific genome editing was recently revolutionized by the
discovery and characterization of a programmable, RNA guided DNA
endonuclease from Streptococcus pyrogenes called Cas9
which operates together with CRISPR (Clustered Regularly Interspaced
Palindromic Repeats) loci in the bacterial genome as a form of defense against
invading plasmids or DNA viruses, in bacteria
CRISPR/Cas9

4. What is CRISPR-cas9 system?
The CRISPR associated protein Cas9 is an endonuclease that uses a guide sequence
within an RNA duplex, tracrRNA:crRNA, to form base pairs with DNA target
sequences, enabling Cas9 to introduce a site-specific double-strand break in the DNA.
•These play a key role in a bacterial defense system, form the basis of a genome
editing technology known as CRISPR-Cas9 that allows permanent modification of
genes within organisms.
A sequence at the 5` side that determines the DNA target site by Watson-Crick base-
pairing and a duplex RNA structure at the 3` side that binds to Cas9.

5. .
Cell. Mol. Life Sci. (2014)

6. CRISPR-CAS9 TECHNOLOGY

7.  While native CRISPR/Cas systems have a variety of enzymes responsible for
processing foreign DNA as well as the RNA guides required for endonuclease
function,
 1.sgRNA(single guide RNA) 2.Cas9
Rotem Sorek and C. Martin Lawrence et al (2013) .Annu. Rev. Biochem. 82:237–66
Key Components
The dual tracrRNA:crRNA was engineered
as a single guide RNA (sgRNA) that
retains two critical features

8. Diagram showing how the CRISPR-Cas9 editing tool works Genome Research Limited

9. Biology of the type II-A CRISPR-Cas system. The type II-A system from S. pyogenes is shown as an
example. (A) The cas gene operon with tracrRNA and the CRISPR array. (B) The natural pathway of
antiviral defense involves association of Cas9 with the antirepeat-repeat RNA (tracrRNA: crRNA)
duplexes, RNA co-processing by ribonuclease III, further trimming, R-loop formation, and target
DNA cleavage. (C) Details of the natural DNA cleavage with the duplex tracrRNA:crRNA
Jennifer A and Emmanuelle Charpentier*, (2016). Science 346 VOL 346 ISSUE 6213

10. Jennifer A and Emmanuelle Charpentier*, (2016). Science 346 VOL 346 ISSUE 6213

11. Syed Shan-e-Ali Zaidi (2016) Front. Plant Sci.,

12. 2. Design your gRNA
1. Choose your target
5. Monitor indels
4. Target your sgRNA and Cas to the cell
3. Test sgRNA efficacy in vitro

13. Lowder L, et al
(2016)
Front. Plant Sci.
7:1683
Delivery of CRISPR Reagents to Plant Cells and Tissues

14. The CRISPR/Cas9 technology has recently become a novel antiviral tool for plants.
(Zhang D etal, Genome editing: Nat Plants 2015)
Using CRISPR/Cas9 technology to introduce sequence-specific deleterious point
mutations at the eIF(iso)4E locus in Arabidopsis thaliana to successfully engineer
complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field-grown
vegetable crops
(Pyott DE et al, Pubmed Oct;17)
Destruct invading viral DNA for virus resistance development in plants was first
reported in 2015 by three independent communications; two in Nature Plants by Ji et al.
& Baltes et al. followed by one in Genome Biology by Ali et al. against geminiviruses
(Ali Z etal, CRISPR/Cas9-Sci Rep 2016)
The CRISPR Approaches to make Virus Resistant Plants

15. The applications of CRISPR/Cas9 mediated virus resistance in plants have been
so far limited mainly to model species demonstration like Tobacco and
Arabidopsis targeting the viral genes responsible for replication
Beet severe curly top virus (BSCTV), Bean yellow dwarf virus (BeYDV),
Tomato yellow leaf curl virus (TYLCV), Beet curly top virus (BCTV), Merremia
mosaic virus (MeMV), Cotton leaf curl Kokhran virus (CLCuKoV), Turnip
mosaic virus (TuMV).
(Zaidi, S. S.-et al Plant Sci. 2016)

16. The CRISPR/Cas9 mediated virus resistance in Nicotiana benthamiana against the
geminivirus, beet severe curly top virus (BSCTV), by introducing mutations at the viral
target sequences
(Surender Khatodia et al February 2017) Taylor and Francis Bioengineered

17. Baltes et al., targeted the replication initiator protein (Rep) gene of bean yellow dwarf
virus (BeYDV) using CRISPR/Cas9 system in transgenic N. benthamiana plants and
introduced mutations which resulted in virus resistance
(Baltes NJ et al Nat Plants 2015)
The applicability of the Cas9/gRNA system for virus resistance development in plants
by targeting the viral Rep, coat proteins genes and the conserved intergenic region (IR)
in N. benthamiana plants against the Tomato yellow leaf curl virus (TYLCV), Beet
curly top virus (BCTV) and Merremia mosaic virus (MeMV).
(Ali Z,et al Genome Biol 2015)

18. Surender Khatodia et al February 2017) Taylor and Francis Bioengineered

19. The first report of non-transgenic virus resistance plants was published in 2016, where
the CRISPR/Cas9 technology have been used for targeted genome editing of the
cucumber plants by creating mutations in the eIF4E gene, which results in development
of resistance against three economically important cucumber viruses of the Potyviridae
family, Cucumber vein yellowing virus (CVYV), Zucchini yellow mosaic virus
(ZYMV)
(Pyott DE et al, Pubmed Oct;17
With the
development of novel delivery methods for CRISPR/Cas9 technology Woo et
al.,developed a DNA-free genome editing method in plants by delivering the
preassembled CRISPR-Cas9 ribonucleoproteins.
(Woo JW,et al, Nat Biotechnol 2015)

20. The purified Cas9 protein and guide RNA were preassembled into the Cas9/gRNA complex
and then transfected into plant protoplasts (Arabidopsis thaliana, tobacco, lettuce and rice);
bread wheat and (Grapevine & Apple) which resulted in successful targeted mutagenesis as
that of naturally occurring genetic variation.
Malnoy M,et al (2016) DNA-Front Plant Science and Liang Z, et al (2017) Nature Communications

21. Virus/viruses Plant Target
Viral/host
GM
Transgene
free
Reference
BSCTV N.
benthamiana
and A.
thaliana
IR, CP, and
Rep
GM Ji et al., 2015
BeYDL N.
benthamiana
LIR and
Rep/RepA
GM Baltes et al., 2015
TYLCV, BCTV, and
MeMV
N.
benthamiana
IR, CP, and
Rep
GM Ali et al., 2015
CLCuKoV, TYLCV
2.3, TYLCSV,
MeMV, BCTVLogan,
BCTVWorland
N.
benthamiana
IR, CP, and
Rep
GM Ali et al., 2016
TuMV A. thaliana Host factor
eIF(iso)4E
Transgene free Pyott et al., 2016
CVYV, PRSMV
ZYMV,
Cucumi sativus Host factor eIF4E Transgene free Chandrasekaran et
al., 2016

22. Examples of crops modified with CRISPR technology
51
CROPS DESCRIPTION
Targeted mutagenesis
REFERNCES
Corn Liang et al. 2014
Rice Targeted mutagenesis Belhaj et al. 2013
Sorghum Targeted gene modification Jiang et al. 2013b
Sweet orange Targeted genome editing Jia and Wang 2014
Tobacco Targeted mutagenesis Belhaj et al. 2013
Wheat Targeted mutagenesis Upadhyay et al. 2013,
Yanpeng et al. 2014
Potato
Soybean
Targeted mutagenesis
Gene editing
Shaohui et al., 2015
Yupeng et al., 2015

25. Introduced pHSN401-A7 into N. benthamiana, and pHSN401-C3 into Arabidopsis.

28. CRISPR/Cas9 mediated virus resistance can be developed in any plant species with
well-established genome sequences against any virus and/or multiple viruses
(Geminiviruses and Potyviruses)
DNA free editing with avoiding off target effects by using direct delivery of
Cas9/gRNA ribonucleoprotein complex could further simplify and advanced through
nanoparticles mediated delivery approach
CRISPR/Cas is a breakthrough technology which opens a new field for plant breeding
through the ability to disrupt virus-host interactions, enhance plant defense genes or
reduce plant virus transmission (e.g., by insects).
Future prospective
Can be used to create high degree of genetic variability at precise locus in the genome of
the crop plants.

29. Can be used to eradicate unwanted species like herbicide resistant weeds, insect
pest.
Potential tool for improving polyploidy crops like potato and wheat
Developing biotic and abiotic resistant traits in crop plants.
It is anticipated that the manipulation of plant virus genomes to express CRISPR/Cas
components together with synthetic biology will lead to the next generation of
agricultural products

30. Future applications in Biomedicine and Biotechnology.

32. In general, the development of the CRISPR/Cas9 field is so fast, that it is hard to
foresee all the potential applications, even in the near future. However, without
doubt, due to CRISPR/Cas9, molecular biology has never been more exciting than
now . It has revolutionized the gene editing technology.
The era of straightforward genome editing raises ethical questions that will need to
be addressed by scientists and society at large. How can we use this powerful tool
in such a way as to ensure maximum benefit while minimizing risks?
Regulatory agencies will also need to consider how best to foster responsible use of
CRISPR-Cas9 technology without inhibiting appropriate research and development.