Targeted Insertion in Nicotiana benthamiana Genomes via Protoplast Regeneration

Insertion of a specific sequence in a targeted region for precise editing is still a major challenge in plants. Current protocols rely on inefficient homology-directed repair or non-homologous end-joining with modified double-stranded oligodeoxyribonucleotides (dsODNs) as donors. We developed a simp...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 2653; p. 297
Main Authors Wu, Fu-Hui, Hsu, Chen-Tran, Lin, Choun-Sea
Format Journal Article
LanguageEnglish
Published United States 2023
Subjects
CRISPR-Cas Systems - genetics
Gene Editing - methods
Nicotiana - genetics
Oligodeoxyribonucleotides
Protoplasts
Targeted insertion
CRISPR
Ribonucleoprotein
Protoplast regeneration
Nucleofection
Protoplast transfection
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Summary:Insertion of a specific sequence in a targeted region for precise editing is still a major challenge in plants. Current protocols rely on inefficient homology-directed repair or non-homologous end-joining with modified double-stranded oligodeoxyribonucleotides (dsODNs) as donors. We developed a simple protocol that eliminates the need for expensive equipment, chemicals, modifications of donor DNA, and complicated vector construction. The protocol uses polyethylene glycol (PEG)-calcium to deliver low-cost, unmodified single-stranded oligodeoxyribonucleotides (ssODNs) and CRISPR/Cas9 ribonucleoprotein (RNP) complexes into Nicotiana benthamiana protoplasts. Regenerated plants were obtained from edited protoplasts with an editing frequency of up to 50% at the target locus. The inserted sequence was inherited to the next generation; this method thus opens the possibility for the future exploration of genomes by targeted insertion in plants.
ISSN:1940-6029
DOI:10.1007/978-1-0716-3131-7_19