20210927 Lin S Epub

Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration

Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration

CRISPR J. 2021 Oct 15; 4(5):752-760. Epub 2021 Sep 27.
doi: 10.1089/crispr.2021.0045

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Hsu CT, Yuan YH, Lin YC, Lin S, Cheng QW, Wu FH, Sheen J, Shih MC, Lin CS

Abstract

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 HindIII 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.