CRISPR/dCas9-Mediated Inhibition of Replication of Begomoviruses

Abstract

<ns2:p>CRISPR/Cas9 system with deactivated or dead nuclease domain of Cas9 (known as dCas9) can also be used as a DNA binding protein to target specific DNA sequences. Similar to zinc fingers and transcription activator like effectors (TALEs), dCas9 can be fused with other effector domains for different purposes such as gene activation, gene repression, modification of epigenetic marks etc. Previously, TALEs were used to target and occupy the most conserved nonanucleotide sequence of Cotton Leaf Curl Virus (CLCuV) with promising results. The purpose of this study is to check the efficiency of dCas9 as DNA binding protein targeting the nonanucleotide sequence of CLCuV to inhibit virus replication. Nicotiana benthamiana was used to evaluate the efficiency of CRISPR/dCas9 system for viral interference. The resistance against virus infection and suppression of replication of the virus was assessed by PCR, RT-PCR and symptoms development on plant leaves in terms of days’ post inoculation (dpi). Expression of the Cas9 and gRNA was quantified using RT-PCR. The results showed partial inhibition of CLCuV replication, lower disease symptoms and virus accumulation as compared to control plants. A comparison of current and previous results using TALEs showed that dCas9 was slightly less efficient to suppress virus replication. Multiplexing of gene editing techniques could be the way forward to engineer virus resistance in plants.</ns2:p>