The CRISPR–Cas9, genome editing approach: a promising tool for drafting defense strategy against begomoviruses including cotton leaf curl viruses

Abstract Clustered regularly interspaced palindromic repeats (CRISPR) and associated Cas9 nuclease (CRISPR-Cas9) systems provide adaptive immunity to prokaryotes against infectious phage particles that can be engineered as a genome-editing tool. Guided by an RNA strand, the class II type II CRISPR-Cas9 system can be employed to provide resistance against plant DNA viruses. Here we describe an efficient CRISPR-Cas9 genome editing system based on simultaneous targeting of the highly conserved intergenic region (IR) of the virus that can provide resistance against Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bur) in Nicotiana benthamiana plants. The data revealed that upon infection, the transgenic plants harboring CRISPR-Cas9 and two gRNAs showed complete resistance against CLCuKoV-Bur/Cotton leaf curl Multan betasatellite (CLCuMB). All efforts failed to find the intact virus in CLCuKoV-Bur/CLCuMB challenged transgenic (OX:Cas9NB:IR) plants using either gene specific PCR primers or CLCuKoV-Bur as a probe in southern blot hybridization. Thus, our results have demonstrated an efficient CRISPR-Cas9 approach to engineer durable resistance against CLCuKoV-Bur in a model system. The implications of these findings are discussed.

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Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus

International Journal of Molecular Sciences ◽

10.3390/ijms222212543 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12543

Author(s):

Barkha Binyameen ◽

Zulqurnain Khan ◽

Sultan Habibullah Khan ◽

Aftab Ahmad ◽

Nayla Munawar ◽

...

Keyword(s):

Genome Editing ◽

Insect Pests ◽

Restriction Analysis ◽

Cotton Leaf ◽

Cotton Production ◽

Colony Pcr ◽

Guide Rna ◽

Cotton Leaf Curl Virus ◽

Leaf Curl Virus ◽

Leaf Curl

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3–5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20–40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60−70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.

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First genome edited poinsettias: targeted mutagenesis of flavonoid 3′-hydroxylase using CRISPR/Cas9 results in a colour shift

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-021-02103-5 ◽

2021 ◽

Author(s):

Daria Nitarska ◽

Robert Boehm ◽

Thomas Debener ◽

Rares Calin Lucaciu ◽

Heidi Halbwirth

Keyword(s):

Transgenic Plants ◽

Genome Editing ◽

Ornamental Plants ◽

Targeted Mutagenesis ◽

Cloning And Expression ◽

Euphorbia Pulcherrima ◽

Loss Of Function ◽

Wild Type ◽

Promising Tool ◽

Reddish Orange

AbstractThe CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3′-hydroxylase (F3′H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar ‘Christmas Eve’, in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3′H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3′H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3′H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3′H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia.

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Implication of the Whitefly Protein Vps Twenty Associated 1 (Vta1) in the Transmission of Cotton Leaf Curl Multan Virus

Microorganisms ◽

10.3390/microorganisms9020304 ◽

2021 ◽

Vol 9 (2) ◽

pp. 304

Author(s):

Yao Chi ◽

Li-Long Pan ◽

Shu-Sheng Liu ◽

Shahid Mansoor ◽

Xiao-Wei Wang

Keyword(s):

Coat Protein ◽

Molecular Mechanisms ◽

Cotton Leaf ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Vacuolar Protein ◽

Leaf Curl Disease ◽

Two Hybrid ◽

Asia Ii 1 ◽

Leaf Curl

Cotton leaf curl Multan virus (CLCuMuV) is one of the major casual agents of cotton leaf curl disease. Previous studies show that two indigenous whitefly species of the Bemisia tabaci complex, Asia II 1 and Asia II 7, are able to transmit CLCuMuV, but the molecular mechanisms underlying the transmission are poorly known. In this study, we attempted to identify the whitefly proteins involved in CLCuMuV transmission. First, using a yeast two-hybrid system, we identified 54 candidate proteins of Asia II 1 that putatively can interact with the coat protein of CLCuMuV. Second, we examined interactions between the CLCuMuV coat protein and several whitefly proteins, including vacuolar protein sorting-associated protein (Vps) twenty associated 1 (Vta1). Third, using RNA interference, we found that Vta1 positively regulated CLCuMuV acquisition and transmission by the Asia II 1 whitefly. In addition, we showed that the interaction between the CLCuMuV coat protein and Vta1 from the whitefly Middle East-Asia Minor (MEAM1), a poor vector of CLCuMuV, was much weaker than that between Asia II 1 Vta1 and the CLCuMuV coat protein. Silencing of Vta1 in MEAM1 did not affect the quantity of CLCuMuV acquired by the whitefly. Taken together, our results suggest that Vta1 may play an important role in the transmission of CLCuMuV by the whitefly.

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