Engineering broad-spectrum resistance to cotton leaf curl disease by CRISPR-Cas9 based multiplex editing in plants

Abstract

Advances in genome editing technologies have tremendous potential to address the limitations of classical resistance breeding. CRISPR-Cas9 based gene editing has been applied successfully in plants to tolerate virus infections. In this study, we successfully tested CRISPR-Cas9 system to counteract cotton leaf curl disease (CLCuD) caused by whitefly transmitted cotton leaf curl viruses (CLCuVs). We also analyzed the ability of CLCuV to escape the Cas9 endonuclease activity. Targeting overlapping genes of most prevalent CLCuVs with three gRNAs resulted in virus interference, as validated by low virus titer. Furthermore, multiplex CRISPR-Cas9 construct simultaneously targeting six genes of CLCuV, was found more effective to interfere with virus proliferation compared to targeting single region individually. Additionally, transgenic N. benthamiana plants expressing multiple gRNAs simultaneously showed enhanced tolerance against CLCuV infection when compared to wild-type plants. T7 Endonuclease-I (T7EI) assay, showing indels in the CLCuV genome, confirmed the occurrence of double strand breaks (DSBs) in DNA at target sequence induced by Cas9 endonuclease. We observed that targeting CLCuV genome at multiple sites simultaneously resulted in better interference, also with inefficient recovery of altered virus molecules. Next, we tested multiplex construct in cotton to interfere CLCuV infection. We found significant decrease in virus accumulation in cotton leaves co-infiltrated with multiplex cassette and virus compared to cotton leaves infiltrated with virus only. The results demonstrate future use of CRISPR-Cas9 system for engineering virus resistance in crops. Moreover, our results also advocate that resistance to mixed virus infections can be engineered using multiplex genome editing.

CRISPR-Cas-Led Revolution in Diagnosis and Management of Emerging Plant Viruses: New Avenues Toward Food and Nutritional Security

“…In a recent study, a transient assay was performed by multiplexing of gRNA-Cas9 modules in the model plant N. benthamiana , and showed a high degree of resistance to chili leaf curl virus (ChiLCV) and restriction of the generation of escape mutants as compared to single sgRNA ( 220 ). A similar study on targeting multiple overlapping viral genes of cotton leaf curl virus (CLCuV) resulted in better interference with virus proliferation ( 221 ). A schematic representation of different usage of the CRISPR-Cas tool indicates its versatile applications right from precise genome editing to diagnosis within a short period ( Figures 2 , 3 ).…”

“…Several independent studies on CRISPR-engineered broad-spectrum disease resistance have demonstrated its versatile applications in various crop species. Moreover, several recent developments have been made to generate CRISPR-engineered dicotyledonous plants by de novo meristem induction ( 220 ), large DNA insertion (up to 2 kb) in rice ( 221 ), enhanced gene targeting efficiency through a heat-inducible CRISPR system in maize ( 222 ), and reconstructing the plant genome through genome engineering and somatic chromosome engineering, enabling genetic linkage ( 223 ). The CRISPR technique has been practically utilized to impart resistance to several plant RNA and DNA viruses.…”

LbCas12a mediated suppression of Cotton leaf curl Multan virus

“…Binyameen et al (2021) have also demonstrated a similar method that showed efficient suppression of CLCuKoV by simultaneously targeting multiple viral genes by multiplexed CRISPR/Cas9 thus reducing virus accumulation in host plants. In another study, Mubarik et al (2021) also determined the virus accumulation in multiplex CRISPR/Cas9 transformed plants through real-time PCR and observed a 60-70% reduction in virus accumulation of CLCuKoV in transgenic plants. In another study, Khan et al (2020) have demonstrated the reduced virus accumulation by 40-80% in benthimiana plants transformed with CRISPR/Cas9 against CLCuKoV.…”

A comprehensive review on Gossypium hirsutum resistance against cotton leaf curl virus

“…The genome component DNA-B (absent in CLCuV but present in bi-partite Begomoviruses ) encode the movement protein (MP) in the complementary-sense orientation and nuclear shuttle protein (NSP) in the virion sense ( Sahu et al, 2014 ). DNA-B has two open reading frames and encodes proteins associated with inter and intracellular movements ( Mubarik et al, 2021 ). An intergenic intronic region exists between ORFs of the virion and complementary-sense regions that contain a hair-pin loop-like structure and cis-acting regulatory elements essential for gene expression.…”

“…This alteration resulted in a condensed cotton plant structure that is ideal for mechanized harvesting in contemporary cotton farming ( Wang et al, 2022 ). Recently, Mubarik et al (2021) demonstrated a successful CRISPR-Cas9 system application to combat CLCuD by targeting overlapping genes of prevalent CLCuVs with three guide RNAs (gRNAs). Utilizing a multiplex CRISPR-Cas9 construct targeting six CLCuV genes proved more effective in inhibiting virus proliferation compared to individual targeting.…”