Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration

Abstract

Precise insertion of DNA sequences into specific genome locations is essential for genome editing. Current Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated protein (Cas) protocols rely on homology-directed repair (HDR). These protocols require laborious vector construction and suffer from low efficiency. Oligo DNA can be used as donor DNA (DD) for precise DNA insertion, or targeted insertion (TI) via nonhomologous end joining (NHEJ) in many species. Here, we report a simple protocol that eliminates the need for expensive equipment and vector construction by using polyethylene glycol (PEG) to deliver non-modified synthetic single-stranded oligo DNA (ssODN) and CRISPR-Cas9 ribonucleoprotein (RNP) into protoplasts. Up to 50.0% targeted insertion was achieved in Nicotiana benthamiana and 13.6% in Rapid Cycling Brassica oleracea (RCBO) without antibiotic selection. Using 60 nt DD that contained 27 nt homologous arms, 6 out of 22 regenerated plants showed TI, and one of them had a precise insertion of 6 bp EcoRI (4.5%) in N. benthamiana. Based on whole-genome sequencing, DD inserted only in the double-strain break (DSB) positions that were induced by the CRISPR-Cas RNP. Importantly, the analysis of T1 progenies indicated that the TI sequences were successfully transmitted into the next generation.

Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications

“…These findings demonstrate the possibility and feasibility of utilizing protoplasts for CRISPR-mediated gene editing, particularly in species with a protracted juvenile phase, heterozygosity, or asexual propagation. Likewise, this strategy could represent the most feasible way to directly apply CRISPR-mediated DNA-free genome editing technologies for improving traits and increasing commercial value, as already experimentally confirmed for food and non-food crops, such as strawberry (Martin-Pizarro et al, 2019;Wilson et al, 2019), potato (Gonzalez et al, 2019;Nicolia et al, 2021;, lettuce (Woo et al, 2015), chicory (De Bruyn et al, 2020;Cankar et al, 2022), Nicotiana tabacum (Lin et al, 2018;Hsu et al, 2019) and Brassica oleracea (Lee et al, 2020;Hsu et al, 2021), and ornamental species, as petunia (Yu et al, 2021b). For these reasons, protoplast transient expression systems represent a promising and valid approach for generating CRISPRedited DNA-free plant material and MS mutant production (Figure 2B).…”