CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts

Abstract

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., “polyplexes” that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Recent Advances in Tomato Gene Editing

“…In addition, RNPs have been delivered in tobacco protoplasts through liposome nanoparticles, achieving editing efficiencies as high as 6.0% [ 99 ]. Moreover, recently, a similar protocol has been devised for the successful delivery of RNPs in maize protoplasts [ 100 ].…”

Appreciating animal induced pluripotent stem cells to shape plant cell reprogramming strategies

“…In the context of animal iPSCs, various DNA-free delivery methods have been developed over the years to overcome integrative approaches, thereby enhancing the clinical applicability of iPSCs in humans ( Borgohain et al , 2019 ). To alleviate regulatory constraints and public opposition towards the production of genetically engineered corps, transient expression, or protein delivery into various plant tissues, protoplasts, or cell suspensions are enjoying increased research attention ( Kocsisova and Coneva, 2023 ; Nagy et al , 2023 ; Nakamura et al , 2023 ). The use of rhabdovirus to transiently deliver expression cassettes ( Ma et al , 2020 ) as well as protein delivery via nanoparticles ( Nagy et al , 2023 ) are options in plants.…”

“…To alleviate regulatory constraints and public opposition towards the production of genetically engineered corps, transient expression, or protein delivery into various plant tissues, protoplasts, or cell suspensions are enjoying increased research attention ( Kocsisova and Coneva, 2023 ; Nagy et al , 2023 ; Nakamura et al , 2023 ). The use of rhabdovirus to transiently deliver expression cassettes ( Ma et al , 2020 ) as well as protein delivery via nanoparticles ( Nagy et al , 2023 ) are options in plants. Direct delivery of proteins into cells theoretically allows for control over dosage and timing of application, and thereby enables the administration of sequential stimulations with different developmental regulators.…”

“…Recently, cell-penetrating peptides (CPPs) were used to deliver WUS protein into Arabidopsis seedlings, where they could move from the cytosol into the nucleus and recapitulate the WUS-specific transcriptional downstream response. However, WUS-mediated somatic embryogenesis was not observed ( Nagy et al , 2023 ), suggesting that different levels of WUS protein or additional factors are required. In light of this, the effects of the repressive WOX13 during shoot induction demonstrate that attention should also be paid to the inhibitory elements in the regeneration process.…”