Vol.:(0123456789) 1 3 Molecular Biology Reports https://doi.org/10.1007/s11033-020-05409-3 ORIGINAL ARTICLE Virus-induced CRISPR-Cas9 system improved resistance against tomato yellow leaf curl virus Parisa Ghorbani Faal 1  · Mohammad Farsi 1  · Alireza Seif 1  · Amin Mirshamsi Kakhki 1 Received: 7 December 2019 / Accepted: 26 March 2020 © Springer Nature B.V. 2020 Abstract Plant viruses are the most signifcant factors associated with massive economical losses in agricultural industries worldwide. Accordingly, many studies are dedicated to making virus-resistant crop varieties each year due to the ever-changing nature of viruses. Recently genome engineering methods have been used to confer interference against eukaryotic viruses. Research results on genome editing technics, in particular, CRISPR-Cas9, promises a feasible solution to make virus-resistant crops. In this research, we explored the possibility of utilizing CRISPR-Cas9 to obtain TYLCV resistant tomato varieties. Moreover, to overcome any potential of-target efects of Cas9, we used an inducible promoter to initiate Cas9 activity in case of the virus attack. Cas9 vector was transformed by the rgsCaM promoter, known as an endogenous silencer of RNAi and overex- pressed after a virus attack. The golden gate cloning method was applied to construct sgRNAs. Intergenic region and coat protein-coding sequences of TYLCV were used to design sgRNAs. Infltrated sensitive Money Maker varieties analyzed by real-time PCR, showed a signifcant reduction or delayed accumulation of viral DNA compared to the control plants. This result demonstrates the efciency of using an inducible promoter in CRISPR-Cas9 constructs. Keywords TYLCV · CRISPR-Cas9 · rgsCaM promoter · Inducible Cas9 · Virus resistance Introduction Tomato Yellow Leaf Curl Virus (TYLCV), a member of the genus Begomovirus, causes signifcant economic losses in tomato production worldwide [1]. TYLCV has a 2.7 kb ssDNA genome [2], encompassing six overlapping open reading frames (ORFs) [3]. Chlorotic leaf margins, thick, rubbery and cupped, small leaves, serious fruit loss, and overall diminish of plants are the common symptoms of infection caused by this virus [4]. Control and manage- ment of the disease caused by TYLCV are laborious and challenging. Earlier methods to control disease focused on removing the viral transmission vector, the silver leaf whitefy (Bemicia tabaci) by insecticides, killing intermedi- ate weeds and changing the cultivation season [5]. Although several resistance genes, including Ty1 or Ty3, are identifed in tomato for TYLCV, due to the genetic linkage between resistance locus and genes related to poor fruit quality, breeding for resistance the virus is challenging [2]. Genetic engineering of tomato by over-expressing the viral capsid protein (cp.) and C4 protein or the non-coding IR sequences to fnd transgenes capable of generating tomato plants with strong resistance to TYLCV has also been challenging [6]. The other approach was based on the idea that binding an artifcial zinc_fnger protein to the Rep binding site or the origin of replication may restrict viral replication. A syn- thetic zinc fnger protein was used to block the replication protein of TYLCV from binding to the origin of replication [7]. Genome editing is becoming a promising technology to mitigate the plant viral disease. The Cas9 protein, derived from type II CRISPR bacterial immune systems, is a pow- erful tool for engineering the genome in diferent organ- isms including plants. Cas9 which is an RNA-guided DNA * Mohammad Farsi mohfarsi@yahoo.com Parisa Ghorbani Faal parisa.ghorbanifaal@um.ac.ir Alireza Seif arseif@um.ac.ir Amin Mirshamsi Kakhki amin.mirshamsi@gmail.com 1 Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran