Open AccessEfficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration
Author(s) -
ChenTran Hsu,
Yasheng Yuan,
YaoCheng Lin,
Steven Lin,
Qiao-Wei Cheng,
Fan Wu,
Jen Sheen,
Ming-Che Shih,
Chun-Shin Lin
Publication year - 2021
Publication title -
the crispr journal
Language(s) - English
Resource type - Journals
eISSN - 2573-1602
pISSN - 2573-1599
DOI - 10.1089/crispr.2021.0045
Subject(s) - genome editing , crispr , oligonucleotide , biology , nicotiana benthamiana , cas9 , plasmid , dna , genome , genome engineering , nuclease , computational biology , insertion , genetics , gene , mutation
Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving CRISPR and Cas proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification, or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60 nt donor containing 27 nt in each homologous arm, 6/22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6 bp Hind III site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.