CRISPRCas9 against virusesAs compared to ZFN or TALEN, CRISPR Cas9is simple, affordable and efficient toolto control viruses (Romay et al.

2017). By using CRISPR-Cas9, antiviraldefense have been developed in plants against Potyviridae and Geminiviridaefamily (Ali et al. 2015, 2016; Baltes et al. 2015;Chandrasekaran et al. 2016; Pyott et al. 2016).

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To deliver sg RNA and Cas9 to plant cells,virus-based vectors are used. For monocots RNA viruses like wheat streak mosaicvirus (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 theyinfect many plant species like wheat, maize, cotton, tomato, cucurbits, beans,legumes and fruits (Nawaz-Ul-Rehman and Fauquet 2009;Rey et al. 2012).Theuse of CRISPR Cas9 as a tool against virus resistance was first reportedagainst Geminiviruses (Ji et al.

2015;Baltes et al. 2015; Ali et al. 2015). They observed that CRISPR Cas9 can reducedisease symptoms in Nicotiana benthamianaagainst bean yellow dwarf virus (BeYDV) and beet severe curly top virus(BSCTV) respectively. When conserved sequence in viral intergenic region istargeted by sgRNA in Nicotianabenthamiana then it provides resistance against tomato yellow leaf curlvirus (TYLCV), beet curly top virus (BCTV) and merremia mosaic virus (MeMV)simultaneously (Ali et al. 2015). Virusresistance is engineered in plants using CRISPR Cas by either modifying hostplant genome or by targeted cleavage of genome of virus (Zaidi et al. 2016).

Also, by delivering preassembled Cas9/gRNAribonucleoprotein complex in plants, can be used as DNA free genome editingtechnique. Targetedcleavage of viral genomeTheCRISPR Cas9 can be used to develop virus resistance in tomato plants and Nicotiana benthamiana by targeting tomato yellow leafcurl virus (TYLCV) genome. The sg RNA targeting CP sequence showed better viralinterference than those targeting the Rep sequence of the TYLCV genome. Also,it is used for developing durable virus resistance as multiple coding andnon-coding virus sequences can be simultaneously targeted (Ali et al.

2015; Tashkandiet al. 2017). CRISPR Cas9 can interfere withGeminivirus by targeting coding and non coding sequences in Nicotiana benthamiana (Ali et al. 2016). It was observed that variants of viruseswere generated when coding sequence was targeted while no viral variantgenerated when non coding intergenic regions were targeted. Thus, targeting noncoding region is necessary for developing durable resistance against multiplebegomoviruses simultaneously like cotton leaf curl kokhran virus (CLCuKoV),tomato yellow leaf curl virus (TYLCV), merremia mosaic virus (MeMV), beet curly top virus (BCTV)-Worland, andbeet curly top virus (BCTV)-Logan. CRISPR Cas9 has been used to eliminate RNAviral infections in Nicotiana benthamianaand Arabidopsis caused due tocucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) by expressing Cas9 ofFrancisella novicida andsgRNA specific for CMV or TMV. It has been observed that the resistance passedon to generations.

Also,the virus accumulation in progenies have reduced (Zhang et al., 2018). One or multiple sg RNA had been deliveredinto Nicotiana benthamiana andArabidopsis thaliana through engineered tobaccorattle virus (TRV) and pea early browning virus (PEBV) where nuclear localization signal have beenoverexpressed containing Cas9. TRV based delivery is less efficient in causingtargeted 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 silicowhich target cotton leaf curl disease (CLCuD)-associated begomovirus inassociation with DNA-satellites. This will provide broad spectrum resistanceagainst viruses. CaMV coat protein (CP) had been targeted using multiple gRNAsin Arabidopsis providing resistance against cauliflower mosaic virus (CaMV)(Liu et al.

2017). CRISPR/Cas9 system has been developedand used for targeted viruses for development of virus resistant plants andhave relevance in crop improvement programmes (Table 1).For successful incorporation of CRISPR/Cas9 system in cropimprovement programs, flexible, well trained and low costs should beconsidered. . Fig. Targeted cleavage ofviral genome using CRISPR Cas9       Table 1.

Use of CRISPR Cas9 system for development ofmultiple 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)    Modificationof host plant genomeForcompletion 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 virusinfection, interaction between eIF4E or eIF(iso)4Eand viral genome-linked protein (VPg) of potyviruses is needed. If eIF4E or eIF(iso)4E is mutated, then it will not interactwith VPg as a result no viral infection takes place. The resistance against turnip mosaic virus (TuMV) in Arabidopsisthaliana 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 virusresistance in cucumber by disrupting function of host recessive eIF4E gene has been reported by Chandrasekaranet al. (2016). This provides resistanceagainst cucumber vein yellowing virus (CVYV), zucchini yellow mosaic virus(ZYMV) and papaya ring spot mosaic virus-W (PRSV-W). This approach thus resultsin broad spectrum resistance against RNA viruses.

Fig. Modificationof host plant genome using CRISPR Cas9 for virus resistance  Delivery of preassembled Cas9/gRNA ribonucleoproteincomplex Nontransgenic plants can be produced without inserting foreign DNA by deliveringpreassembled 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 oftransgenic crops, the transgene can be eliminate from improved varietyCas9/gRNA complex which lead to targeted mutagenesis of upto 46%. The plantshave cell wall present outside the cell membrane due to which nucleic acid orproteins cannot be delivered by techniques like electroporation, transfectionand Agrobacterium-mediated T-DNA delivery. So, to overcome this, cell wall hasbeen removed by digesting it with enzymes.

The protoplast obtained is then usedfor delivery of RNP and editing of genome (Svitashev et al. 2016). DNA vectors have been used to deliver Cas9and sgRNA by particle bombardment into immature wheat embryos. No selectablemarker have been used (Zhang et al.

2016).The plants were regenerated in period of 6-8 weeks. They also found that ontranscribing Cas9 and sgRNA in vitro, the RNA is delivered and is notintegrated in plant’s genome. Thus, mutants containing no transgene isobtained.

Similarly, immature maize embryos were targeted by delivering Cas9and sgRNA with the help of particle bombardment. Again, regenerated plantsdonot 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 targeteffects. Also, it is transgene free, so there is no need for gene segregationthrough backcrossing. This saves lot of time especially in case of crops havingcomplex genome like maize and wheat (Wolter and Puchta, 2017). Fig.Delivery of preassembled Cas9/gRNAribonucleoprotein complex