Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants

Abstract

Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.

Soluble Human Angiotensin- Converting Enzyme 2 as a Potential Therapeutic Tool for COVID-19 is Produced at High Levels In Nicotiana benthamiana Plant With Potent Anti-SARS-CoV-2 Activity

“…The targeting of the protein to the ER provided a high level of production of a number of complex and difficult to-express proteins in N. benthamiana plant, such as full length Pfs48/45 or Pfs230 of P. falciparum ( Farrance et al, 2011 ; Mamedov et al, 2019b ), heptameric form of protective antigen of B. anthracis , human Furin and Factor IX ( Mamedov et al, 2019a ), and RBD of spike protein of SARS-CoV-2 ( Mamedov et al, 2021 ); human Interleukin 6 ( Nausch et al, 2012 ); hemagglutinin from a range influenza viruses ( Chichester et al, 2009 ); monoclonal antibodies ( Vézina et al, 2009 ; Holtz et al, 2015 ); HIV gp140 ( Margolin et al, 2020a ). In addition, other factors that can provide more target accumulation are Agro optimization ( Shamloul et al, 2014 ; Hanittinan et al, 2020 ; Kaur et al, 2021 ), especially using appropriate target specific Agrobacterium strains ( Norkunas et al, 2018 ), activation of agrobacteria with MES (2-morpholinoethanesulfonic acid), MgCl 2 , Acetosyringone ( Shamloul et al, 2014 ; Kaur et al, 2021 ), and to control humidity, temperature, and light during plant growth ( Shamloul et al, 2014 ). In general, these factors allowed the production of the recombinant soluble form of ACE2 at a high level in the N. benthamiana plant.…”

In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components

“…Nevertheless, the prerequisite of protoplast-based assays is the establishment of optimized protoplast isolation procedures for the crop of interest and purified Cas-sgRNA RNP complexes, which further adds to the cost. Transient agroinfiltration assay proved to be a comparatively quick in vivo method for analyzing sgRNA efficacy, but the lower activity and inconsistency depending on target plant species are the significant obstacles [57]. The IRI-CCE platform may serve as a fast way to validate some key aspects.…”

Functional Allele Validation by Gene Editing to Leverage the Wealth of Genetic Resources for Crop Improvement

“…Agroinfiltration is the most common form of leaf infiltration, which leads to the transfer of single-stranded T-DNA from Agrobacterium to the plant cells [ 163 ]. Agroinfiltration can be achieved by different methods, such as syringe infiltration, vacuum infiltration, hydrogen peroxide-based agroinfiltration, leaf disc vacuum infiltration, spray-based agroinfiltration, and the detached leaf-based infiltration approach [ 164 , 165 , 166 , 167 , 168 ]. Agroinfiltration has been used with the CRISPR/Cas9 system for transient delivery of Cas9 and sgRNA for targeted gene mutations in plants [ 169 ].…”