Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application.

Fu-Hui Wu,Qiao-Wei Cheng,Yu-Hsuan Yuan,Jin-Ling Yuan,Choun-Sea Lin,Chen-Tran Hsu,Jin-Jun Yue

doi:10.3389/fgeed.2021.717017

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.

Highlights

Many genes and single-nucleotide polymorphisms (SNPs) related to important phenotypes have been identified by an array of bioinformatic tools utilizing the rich and diverse genome resources currently available (Varshney et al, 2009)
We developed a simple protocol, the Tape-Arabidopsis Sandwich, in which the lower epidermal layer of an Arabidopsis thaliana (Arabidopsis) leaf is removed with regular office tape to expose mesophyll cells to cell-wall-digesting enzymes (Wu et al, 2009)
There are approximately 2,000 articles referring to the use of the Transient Expression in Arabidopsis Mesophyll Protoplast (TEAMP) system for basic research (Yoo et al, 2007), and we have developed and optimized isolation and transfection protocols for important crops (Lin et al, 2018)

Summary

INTRODUCTION

Many genes and single-nucleotide polymorphisms (SNPs) related to important phenotypes have been identified by an array of bioinformatic tools utilizing the rich and diverse genome resources currently available (Varshney et al, 2009). CRISPR-mediated, DNA-free genome editing in protoplasts followed by regeneration into whole plants would be the most feasible way to directly apply gene editing technologies to improve traits and increase commercial value This method has already been experimentally proven in protoplasts including potato (Andersson et al, 2017; Andersson et al, 2018; Tuncel et al, 2019; González et al, 2020; Zhao et al, 2021; Nicolia et al, 2021), N. tabacum (Lin et al, 2018; Hsu et al, 2019), N. benthamiana (Hsu et al, 2021a,b), Brassica oleracea Another group has achieved this feat for lettuce (Woo et al, 2015)

DISCUSSION

DATA AVAILABILITY STATEMENT