Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts

Abstract

The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or three RNPs were delivered simultaneously, long deletions of 33–152 nt between the gRNA target sites were generated. Such mutations occurred with an efficiency of up to 12%, while the overall editing effectiveness was very high, reaching 71%. This highly efficient multiplex gene-editing method, without the need for recombinant DNA technology, can be adapted to other plants for which protoplast culture methods have been established.

Non-GM Genome Editing Approaches in Crops

“…The use of CRISPR/Cas9 RNPs was first reported in 2014, for human cell mutagenesis ( Kim et al, 2014 ), and have since been extensively adopted for plant genome editing in a variety of plant species including Arabidopsis thaliana , rice, lettuce, tobacco ( Woo et al, 2015 ; Kim et al, 2017 ), petunia ( Subburaj et al, 2016 ; Yu et al, 2021 ), grapevine, apple ( Malnoy et al, 2016 ), maize ( Svitashev et al, 2016 ), wheat ( Liang et al, 2017 ; Liang et al, 2018 ), soybean ( Kim et al, 2017 ), potato ( Andersson et al, 2018 ; González et al, 2020 ; Nicolia et al, 2021b ), cabbage ( Murovec et al, 2018 ; Park et al, 2019 ; Lee et al, 2020 ), banana ( Wu et al, 2020 ), pepper ( Kim et al, 2020 ), witloof ( De Bruyn et al, 2020 ), carrot ( Klimek-Chodacka et al, 2021 ), and tomato ( Nicolia et al, 2021a ). In most cases, polyethylene glycol-calcium (PEG-Ca 2+ )-mediated cell transfection was the method used to deliver the RNPs into plant protoplasts.…”

Advances in Delivery Mechanisms of CRISPR Gene-Editing Reagents in Plants

“…Once efficient protoplast isolation, transfection, and regeneration have been established in a plant species, it could be a high throughput platform by combining with flow cytometry and omic analyses for optimizing gene-editing. Furthermore, multiplexing, editing multiple genes at a time has been achieved using protoplasts ( Klimek-Chodacka et al, 2021 ; Nicolia et al, 2021 ; Yu et al, 2021 ; Zhang et al, 2021 ). By co-delivering Three Prime Repair exonuclease 2 (TREX2) and CRISPR/Cas9 into protoplasts, targeted mutagenesis using a multiplexing strategy was further improved ( Weiss et al, 2020 ) ( Figure 2A ).…”