AUTHOR=Yue Jin-Jun , Yuan Jin-Ling , Wu Fu-Hui , Yuan Yu-Hsuan , Cheng Qiao-Wei , Hsu Chen-Tran , Lin Choun-Sea TITLE=Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application JOURNAL=Frontiers in Genome Editing VOLUME=3 YEAR=2021 URL=https://www.frontiersin.org/journals/genome-editing/articles/10.3389/fgeed.2021.717017 DOI=10.3389/fgeed.2021.717017 ISSN=2673-3439 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.