Genome editing is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or "molecular scissors”. Identified CRISPR-associated protein 9 from Streptococcus pyogenes (Cas9) in (2012). Genome engineering by CRISPR/Cas system (16 February 2012). New application of this technology to combating viral infection by destroying invading virus DNA has now become possible in plants.

1. CRISPR- Cas technology-
A new antiviral weapon for plants
Presentation by:
Bhor Sachin Ashok (PhD, III year)
Ehime University, Matsuyama, JAPAN

2. Genome editing is a type of genetic engineering in which DNA is
inserted, replaced, or removed from a genome using artificially
engineered nucleases, or "molecular scissors”.
Introduction
Genome editing tools :
1) Zinc finger nucleases (ZFNs):
2) Transcription activator–like effector nucleases (TALENs):
3) CRISPR-Clustered Regularly Interspaced Short Palindromic Repeats

3. “A journey from bacterial CRISPR immune
system to engineered RNA Guided Nucleases
(RGNs)”…
 First described (1987)
 48% bacteria; 84% archaea
 Short Regularly Spaced Repeats (SRSR)
(2000)
 Renamed CRISPR (2002)
 A prokaryotic adaptive immune system
(2005-2012)
 Identified CRISPR-associated protein 9 from
Streptococcus pyogenes (Cas9) in (2012)
 Genome engineering by CRISPR/Cas
system (16 February 2012)
 CRISPR Interference (2013)
 Other applications of CRISPR (2013- till
date)
CRISPR-
Clustered Regularly Interspaced Short Palindromic Repeats
(www.the-scientist.com )

4. The most widely used engineered CRISPR-Cas system

5. An Overview of the applications of CRISPR- Cas
system
Fig. 3. An overview of the applications of CRISPR-cas9 system in
functional genomics. CRISPR-Cas9 system can be used for genome
editing (via introduction of point mutations, insertions or deletions),
transcriptional regulation (via CRISPR interference, activation, repression
or epigenetic modulation) or Forward genetic screens (via generation of
loss of function, knock down or activation mutants using sgRNA libraries.

6.  Plant viruses cause enormous losses in worldwide crop
production every year.
 The alternate solution is to engineer enhanced virus resistance in
crops through -
1) RNA interference (RNAi)
2) artificial microRNAs (amiRNAs)
 New application of this technology to combating viral infection by
destroying invading virus DNA has now become possible in
plants.
Application of CRISPR- Cas technology in virus
resistance to plants

7. CRISPR- Cas-based geminivirus destruction
Figure. 1. Schematic of CRISPR- Cas-based geminivirus destruction in a plant
cell. Invading geminivirus DNA (blue circle) replicates in the plant nucleus via
rolling circle replication. The Cas9- sgRNA complex acts as a molecular scissor to
cleave viral DNA and block this replication (top). Additionally, the Cas9–sgRNA
complex could potentially cause unwanted off-target cleavage in the plant genome
in addition to virus cleavage (bottom).

9. BSCTV genome structure and design of the sgRNA
target sites
Figure S1. BSCTV genome structure and design of the sgRNA target sites. (a) The
schematic of the BSCTV genome shows the positions of the seven openreading
frames (V1, V2, V3 on the sense strand and C1, C2, C3, C4 on the complementary
sense strand). Regions A (in the intergenic region), B (in V1) and C (in C1), each
consisting of about 300 bp, were selected for sgRNA target sequence design. (b)
Positions of the 43 sgRNA target sites in the target regions: 13 target sites in region
A, 15 target sites in region B and 15 target sites in region C.

10. Figure 1a. The overview of sgRNA–Cas9-based sequence-specific
system for conferring geminivirus resistance in plants.

11. Figure S2. Transient transformation system for selecting highly active sgRNAs
in N. benthamiana. Thirty-day-old N. benthamiana plants with between 6 and 8
true leaves were injected with A. tumefaciens strain EHA105 harbouring
PHSN401-sgRNA vector resuspended to a final OD600 of 1.5. Two days later,
A. tumefaciens strain EHA105 harboring pCambia1300-BSCTV vector was
resuspended to a final OD600 of 0.5 and injected into the previously infected
leaves.

12. Figure S3. Confirmation that the sequence-specific sgRNA/Cas9 system (A7, B7
and C3) cuts replicating virus. (a) The structure of experimental and mock vectors
used in the transient assay. (b-e) Quantification of the antivirus activity of these
constructs. Viral accumulation was measured by qPCR. Error bars represent SD.

13. Fig. 2b. The activities of 43 sgRNAs
targeting the BSCTV genome. Red
arrows indicate the systemic leaves
with different phenotypes after BSCTV
infection. Each region has sgRNAs
conferring strong resistance (left
photograph) and others conferring mild
resistance (middle photograph)
compared with WT (right photograph).
WT represents the pHSN401 vector.
Scale bar, 2 cm.

14. Fig. 2C. Confirmation that the sequence-specific sgRNA–Cas9 system cuts
replicating virus. A leaf from a 30-day-old N. benthamiana plant was marked with
four circular regions. The pCambia-BSCTV construct was injected into the top part of
the leaf and one of the experimental vectors and the control vector were injected into
separate areas of the bottom part of the leaf. Six days after injection, the DNA in the
bottom regions (marked by dashed lines) was extracted and viral accumulation was
measured by qPCR (as shown by the bar charts). Error bars represent s.d., asterisks
indicate significance *P < 0.05, **P < 0.01.

15. Figure 2d T7E1 assay detecting sgRNA–Cas9-induced mutations in the
BSCTV genome. Red arrowheads indicate fragments generated by T7E1.
and 2e) sgRNA–Cas9-induced mutations at the targeting sites. −/+ indicates
deletion/ insertion of nucleotides.

16. Figure S4. Inoculation of transgenic Arabidopsis and N. benthamiana plants with
BSCTV. For Arabidopsis, when plants had been grown for about one month with
their primary inflorescences less than 1-2cm, these inflorescences were cut off
and A. tumefaciens strain GV3101 harboring pCambia1300-BSCTV vector,
resuspended to a final OD600 of 1.5, was injected into the stem. For N.
benthamiana, the A. tumefaciens strain EHA105 harboring pCambia1300-BSCTV
vector resuspended to a final OD600 of 0.2 was injected directly into the leaves.

17. Figure 3a. Transgenic N. benthamiana and Arabidopsis plants develop resistance to
BSCTV. Transgenic N. benthamiana and Arabidopsis plants develop resistance to
BSCTV. a, d, The symptoms of transgenic and WT N. benthamiana (a), transgenic
and WT Arabidopsis (d) after virus inoculation. Scale bar, 2 cm (a) and 3 cm (d).

18. Figure 3b,e, Expression levels of Cas9 and sgRNA in transgenic N. benthamiana (b)
and transgenic Arabidopsis (e). Error bars represent s.d. *P < 0.05. c,f, Virus
accumulation in N. benthamiana (c) and Arabidopsis (f) assessed by Southern blots.
OC, open circle double-stranded DNA; SS, single-stranded DNA; SC, supercoiled
double-stranded DNA; sg, subgenomic forms of virus DNA.

20. Figure 4a. Testing Cas9-sgRNA activity at targets within the BeYDV. a, Illustration of
the wild type BeYDV genome (reverse complemented) and sgRNA target sequences.
Red nucleotides, sgRNA sequence; black nucleotides, BeYDV sequence; blue
nucleotides, protospacer adjacent motif; lightning bolts, predicted sites for Cas9
cleavage; IR, inverted repeat; 9 nt, nanonucleotide sequence; M1, motif 1; M2, motif
2; M3, motif 3; +/−, DNA strand that pairs with the sgRNA; NOS, nopaline synthase.

21. Figure 4b. Approach to assess Cas9 and sgRNA activity within N. benthamiana
leaves by measuring GFP expression.

22. Figure S1: Comparison of different CRISPR-Cas architectures for reducing
virus expression. All experimental samples were transformed within a single
leaf. Error bars represent standard deviation of 10 biological replicates.
Experimental samples were normalized to their respective gTM3+ control.
Asterisks, P<0.0005

23. Figure 5. a. Restricting BeYDV infection with the CRISPR–Cas system. a, Illumina
next-generation sequencing of four sgRNA targets. Dark bars, sgRNA gTM3+
(negative control). b, PCR detection of deletions within the genome of mobile
BeYDV. The ∼657 bp band indicates unmodified sequences or sequences with
small INDELs. The ∼349 bp band indicates sequences containing deletions;
sequences of three clones for each biological replicate are shown.

24. Figure 5c,d. Representative images of transgenic plants challenged with BeYDV
35 days after inoculation. d, Quantitative PCR of virus levels within transgenic
plants 37 days after inoculation.

25. Thank you…!!!