Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application

Abstract

In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.

Keywords: CRISPR/cas (clustered regularly interspaced short palindromic repeats); DNA-free; RNP; protoplasts; transient transfection.

FIGURE 1

Protoplast regeneration and CRISPR genome editing. (A) Protoplast regeneration-related articles according to taxonomy. These 779 protoplast regeneration articles were identified from Google scholar and NCBI (Supplementary Table S1). The taxonomy of the plant species follows the Angiosperm Phylogeny Group IVsystem and NCBI taxonomy. Major familes are color coded as follows: purple; Asteraceae, light orange; Solanaceae, green; Brassicaceae, orange; Rutaceae, blue; Fabaceae, yellow-green; Poaceae. The names of the species reported in more than five articles are shown. The rest of the species are shown in grey. The information used to create this diagram is shown in Supplementary Table S1. (B) Schematic of tobacco protoplast regeneration. Delivery methods stated in grey are the methods used for protoplast transformation that can theoretically be applied in genome editing. The multiple peaks (black arrows in “Efficiency assessment” and “Progeny genotyping”) in the upstream of PAM (green boxes) are indicated. tThe target site (red boxes) is editied by CRISPR reagent.