CRISPR
Cas9 against viruses

As compared to ZFN or TALEN, CRISPR Cas9
is simple, affordable and efficient tool
to control viruses (Romay et al. 2017). By using CRISPR-Cas9, antiviral
defense have been developed in plants against Potyviridae and Geminiviridae
family (Ali et al. 2015, 2016; Baltes et al. 2015;
Chandrasekaran et al. 2016; Pyott et al. 2016). To deliver sg RNA and Cas9 to plant cells,
virus-based vectors are used. For monocots RNA viruses like wheat streak mosaic
virus (WSMV) and barley stripe mosaic virus(BSMV) (Lee et al. 2012) are used and for dicots tobacco rattle virus
(TRV) is used. Geminiviruses are also used as vectors in many crops as they
infect many plant species like wheat, maize, cotton, tomato, cucurbits, beans,
legumes and fruits (Nawaz-Ul-Rehman and Fauquet 2009;
Rey et al. 2012).

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The
use of CRISPR Cas9 as a tool against virus resistance was first reported
against Geminiviruses (Ji et al. 2015;
Baltes et al. 2015; Ali et al. 2015). They observed that CRISPR Cas9 can reduce
disease symptoms in Nicotiana benthamiana
against bean yellow dwarf virus (BeYDV) and beet severe curly top virus
(BSCTV) respectively. When conserved sequence in viral intergenic region is
targeted by sgRNA in Nicotiana
benthamiana then it provides resistance against tomato yellow leaf curl
virus (TYLCV), beet curly top virus (BCTV) and merremia mosaic virus (MeMV)
simultaneously (Ali et al. 2015). Virus
resistance is engineered in plants using CRISPR Cas by either modifying host
plant genome or by targeted cleavage of genome of virus (Zaidi et al. 2016). Also, by delivering preassembled Cas9/gRNA
ribonucleoprotein complex in plants, can be used as DNA free genome editing
technique.

Targeted
cleavage of viral genome

The
CRISPR Cas9 can be used to develop virus resistance in tomato plants and Nicotiana benthamiana by targeting tomato yellow leaf
curl virus (TYLCV) genome. The sg RNA targeting CP sequence showed better viral
interference than those targeting the Rep sequence of the TYLCV genome. Also,
it is used for developing durable virus resistance as multiple coding and
non-coding virus sequences can be simultaneously targeted (Ali et al. 2015; Tashkandi
et al. 2017). CRISPR Cas9 can interfere with
Geminivirus by targeting coding and non coding sequences in Nicotiana benthamiana (Ali et al. 2016). It was observed that variants of viruses
were generated when coding sequence was targeted while no viral variant
generated when non coding intergenic regions were targeted. Thus, targeting non
coding region is necessary for developing durable resistance against multiple
begomoviruses simultaneously like cotton leaf curl kokhran virus (CLCuKoV),
tomato yellow leaf curl virus (TYLCV), 
merremia mosaic virus (MeMV), beet curly top virus (BCTV)-Worland, and
beet curly top virus (BCTV)-Logan. CRISPR Cas9 has been used to eliminate RNA
viral infections in Nicotiana benthamiana
and Arabidopsis caused due to
cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) by expressing Cas9 of
Francisella novicida and
sgRNA specific for CMV or TMV. It has been observed that the resistance passed
on to generations. Also,
the virus accumulation in progenies have reduced (Zhang et al., 2018). One or multiple sg RNA had been delivered
into Nicotiana benthamiana and
Arabidopsis thaliana through engineered tobacco
rattle virus (TRV) and pea early browning virus (PEBV) where nuclear localization signal have been
overexpressed containing Cas9. TRV based delivery is less efficient in causing
targeted mutation than PEBV (Ali et al. 2017).
The use of CRISPR/Cas9 system in cotton against cotton leaf curl disease
(CLCuD) has been reported by Iqbal et al. (2016).
They designed multiple gRNAs in silico
which target cotton leaf curl disease (CLCuD)-associated begomovirus in
association with DNA-satellites. This will provide broad spectrum resistance
against viruses. CaMV coat protein (CP) had been targeted using multiple gRNAs
in Arabidopsis providing resistance against cauliflower mosaic virus (CaMV)
(Liu et al. 2017). CRISPR/Cas9 system has been developed
and used for targeted viruses for development of virus resistant plants and
have relevance in crop improvement programmes (Table 1).
For successful incorporation of CRISPR/Cas9 system in crop
improvement programs, flexible, well trained and low costs should be
considered.

. Fig. Targeted cleavage of
viral genome using CRISPR Cas9

 

 

 

 

 

 

 

Table 1. Use of CRISPR Cas9 system for development of
multiple viral resistance in plants

 

Virus

Target

Plants

References

bean
yellow dwarf virus(BeYDV), beet severe curly top virus
(BSCTV)

 Viral DNA

Nicotiana
benthamiana

Ji
et al. (2015)
 Baltes et al. (2015)
 

tomato
yellow leaf curl virus (TYLCV), beet curly top virus(BCTV) and merremia
mosaic virus (MeMV)

Viral
DNA

Nicotiana
benthamiana

  Ali et al. (2015)

tomato
yellow leaf curl virus (TYLCV)

Viral
DNA

Nicotiana
benthamiana,
tomato

  Ali et al. (2015),
Tashkandi et al.  (2017)

cotton
leaf curl kokhran virus(CLCuKoV), tomato yellow leaf curl virus(TYLCV),  merremia mosaic virus  (MeMV),  beet curly top virus (BCTV)-Worland, and  beet curly top virus (BCTV)-Logan

Viral
DNA

Nicotiana
benthamiana

Ali
et al. (2016)

cotton
leaf curl disease (CLCuD)

Viral
DNA

Cotton

Iqbal
et al. (2016)

cauliflower
mosaic virus (CaMV)

Viral
DNA

Arabidopsis
thaliana

Liu
et al. (2017)

cucumber
mosaic virus (CMV), tobacco mosaic virus (TMV)

Viral
RNA

Arabidopsis
thaliana,  Nicotiana benthamiana
 

Zhang et al. (2018)

Turnip
mosaic virus (TuMV)

Host
genome (eIF(iso) 4E)

Arabidopsis
thaliana

Pyott
et al. (2016)

Cucumber vein yellowing virus (CVYV),  Zucchini yellow mosaic virus (ZYMV),  Papaya ring spot mosaic virus-W (PRSV-W)

Host
genome(eIF4E)

Cucumber

Chandrasekaran et al. (2016)

 

 

 

Modification
of host plant genome

For
completion of life cycle, plant RNA viruses depends on host factors like eukaryotic translation initiation factors eIF4E and eIF(iso)4E (Lellis et al. 2002; Nicaise et al. 2003; Ruffel et al. 2006).
So by disrupting plant genes, viral resistance can be induced. For virus
infection, interaction between eIF4E or eIF(iso)4E
and viral genome-linked protein (VPg) of potyviruses is needed. If eIF4E or eIF(iso)4E is mutated, then it will not interact
with VPg as a result no viral infection takes place. The resistance against turnip mosaic virus (TuMV) in Arabidopsis
thaliana has been developed with the use of CRISPR Cas 9 system by inducing sequence specific point mutation at eIF(
iso) 4E locus (Pyott et al. 2016). The virus
resistance in cucumber by disrupting function of host recessive eIF4E gene has been reported by Chandrasekaran
et al. (2016). This provides resistance
against cucumber vein yellowing virus (CVYV), zucchini yellow mosaic virus
(ZYMV) and papaya ring spot mosaic virus-W (PRSV-W). This approach thus results
in broad spectrum resistance against RNA viruses.

Fig. Modification
of host plant genome using CRISPR Cas9 for virus resistance

 

 

Delivery of preassembled Cas9/gRNA ribonucleoprotein
complex

Non
transgenic plants can be produced without inserting foreign DNA by delivering
preassembled Cas9/gRNA ribonucleoprotein (RNP) complex in plants. In this,
either viral genome or plant genes are targeted. Keeping in view the public and political concerns about the use of
transgenic crops, the transgene can be eliminate from improved variety
Cas9/gRNA complex which lead to targeted mutagenesis of upto 46%. The plants
have cell wall present outside the cell membrane due to which nucleic acid or
proteins cannot be delivered by techniques like electroporation, transfection
and Agrobacterium-mediated T-DNA delivery. So, to overcome this, cell wall has
been removed by digesting it with enzymes. The protoplast obtained is then used
for delivery of RNP and editing of genome (Svitashev et al. 2016). DNA vectors have been used to deliver Cas9
and sgRNA by particle bombardment into immature wheat embryos. No selectable
marker have been used (Zhang et al. 2016).
The plants were regenerated in period of 6-8 weeks. They also found that on
transcribing Cas9 and sgRNA in vitro, the RNA is delivered and is not
integrated in plant’s genome. Thus, mutants containing no transgene is
obtained. Similarly, immature maize embryos were targeted by delivering Cas9
and sgRNA with the help of particle bombardment. Again, regenerated plants
donot contain selectable marker and the efficiency of mutant alleles is 2.4-
9.7% (Svitashev et al. 2016).

This technique has been used in Arabidopsis thaliana, tobacco, lettuce,
rice, bread, wheat, Grapevine and Apple, Petunia (Woo et al. 2015; Malnoy et al. 2016; Subburaj et al. 2016; Liang et al. 2017).
It is advantageous over other methods as there is reduction in off target
effects. Also, it is transgene free, so there is no need for gene segregation
through backcrossing. This saves lot of time especially in case of crops having
complex genome like maize and wheat (Wolter and Puchta, 2017).

Fig.
Delivery of preassembled Cas9/gRNA
ribonucleoprotein complex