A New Protocol for Targeted insertion using CRISPR-Cas9, Oligo Single-Stranded DNA and Protoplast Regeneration

Hsu, C.-T.; Yuan, Y.-H.; Lin, Y.-C.; Lin, S.; Cheng, Q.-W.; Wu, F.-H.; Sheen, J.; Shih, M.-C.; Lin, C.-S.

doi:10.1101/2021.03.09.434087

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bioRxiv · 10.1101/2021.03.09.434087

A New Protocol for Targeted insertion using CRISPR-Cas9, Oligo Single-Stranded DNA and Protoplast Regeneration

Hsu, C.-T.·Yuan, Y.-H.·Lin, Y.-C.·Lin, S.·Cheng, Q.-W.·Wu, F.-H.·Sheen, J.·Shih, M.-C.·Lin, C.-S.

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Abstract

Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (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 50.0% 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 of 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.

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Hsu, C.-T., Yuan, Y.-H., Lin, Y.-C., Lin, S., Cheng, Q.-W., Wu, F.-H., Sheen, J., Shih, M.-C., Lin, C.-S.. 2021-03-10. A New Protocol for Targeted insertion using CRISPR-Cas9, Oligo Single-Stranded DNA and Protoplast Regeneration. https://doi.org/10.1101/2021.03.09.434087

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