WebJan 1, 2024 · The knockout of these selected genes in the chickpea protoplast using DNA-free CRISPR/Cas9 editing represents a novel approach for achieving targeted … WebMar 23, 2024 · Transgene-free genome edited plants can be produced using CRISPR-Cas9 or similar techniques by either developing a transgenic plant followed by outcrossing of the integrated DNA, or by using a transient system as for example protoplast transfection or microprojectile bombardment.

Protoplasts: From Isolation to CRISPR/Cas Genome Editing ... - PubMed

PEG-mediated RNP transfections were performed as previously described18. Briefly, to introduce DSBs using an RNP complex, 1 × 105 protoplast cells were transfected with Cas9 protein (10–60 μg) premixed with in vitro–transcribed sgRNA (20–120 μg). Prior to transfection, Cas9 protein in storage buffer (20 mM … See more Cas9 protein tagged with a nuclear localization signal was purchased from ToolGen, Inc. (South Korea). Templates for guide RNA transcription were generated by oligo-extension using Phusion polymerase … See more To regenerate whole plants, proliferated and elongated adventitious shoots were transferred to a fresh regeneration medium and incubated for 4–6 weeks at 25 °C in the light (16 … See more Protoplasts were isolated as previously described from Arabidopsis18, rice19 and lettuce20. Initially, Arabidopsis (Arabidopsis … See more RNP-transfected protoplasts were re-suspended in 0.5× B5 culture medium23 supplemented with 375 mg/l CaCl2•2H2O,18.35 mg/l NaFe-EDTA, 270 mg/l sodium succinate, 103 g/l sucrose, 0.2 mg/l 2,4 … See more WebNov 5, 2024 · Our study aimed to develop a protocol for DNA-free genome editing of different species of the genus Brassica. Due to their high economic value, we chose cabbage and Chinese cabbage as representatives of the species B. oleracea and B. rapa, respectively, and rapeseed as representative of B. napus. tract of unenclosed uncultivated upland

Establishment of a DNA-free genome editing and protoplast regeneration

WebMar 4, 2024 · In a recent issue of Plant Physiology, Hsu et al. (2024) show a proof-of-concept strategy using protoplast regeneration protocol to generate CRISPR/Cas9-edited allotetraploid wild tomato ( Solanum peruvianum ), an important genomic resource for tomato research and introgression breeding. WebNov 8, 2024 · Heritable DNA-free genome editing of canola ( Brassica napus L.) using PEG-mediated transfection of isolated protoplasts Introduction. Gene editing, which … tract of unenclosed ground