Targeted Gene Knockouts by Protoplast Transformation in the Moss Physcomitrella patens.

Lei Zhu

doi:10.3389/fgeed.2021.719087

Abstract

Targeted gene knockout is particularly useful for analyzing gene functions in plant growth, signaling, and development. By transforming knockout cassettes consisting of homologous sequences of the target gene into protoplasts, the classical gene targeting method aims to obtain targeted gene replacement, allowing for the characterization of gene functions in vivo. The moss Physcomitrella patens is a known model organism for a high frequency of homologous recombination and thus harbors a remarkable rate of gene targeting. Other moss features, including easy to culture, dominant haploidy phase, and sequenced genome, make gene targeting prevalent in Physcomitrella patens. However, even gene targeting was powerful to generate knockouts, researchers using this method still experienced technical challenges. For example, obtaining a good number of targeted knockouts after protoplast transformation and regeneration disturbed the users. Off-target mutations such as illegitimate random integration mediated by nonhomologous end joining and targeted insertion wherein one junction on-target but the other end off-target is commonly present in the knockouts. Protoplast fusion during transformation and regeneration was also a problem. This review will discuss the advantages and technical challenges of gene targeting. Recently, CRISPR-Cas9 is a revolutionary technology and becoming a hot topic in plant gene editing. In the second part of this review, CRISPR-Cas9 technology will be focused on and compared to gene targeting regarding the practical use in Physcomitrella patens. This review presents an updated perspective of the gene targeting and CRISPR-Cas9 techniques to plant biologists who may consider studying gene functions in the model organism Physcomitrella patens.

Highlights

Gene targeting mediated by homologous recombination, widely applied in mouse embryonic stem cells, is useful for studying gene functions
Many genome editing tools have been deployed to obtain knockouts, including nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA-guided CRISPR-Cas family, and gene targeting mediated by endogenous homology-directed repair (HDR) (Capecchi, 2005b; Miller et al, 2007; Sander et al, 2011; Wood et al, 2011; Cong et al, 2013; Knott and Doudna, 2018)
The moss Physcomitrella patens is a unique model organism harboring a high frequency of homologous recombination

Summary

INTRODUCTION

Gene targeting mediated by homologous recombination, widely applied in mouse embryonic stem cells, is useful for studying gene functions. This invention won the Nobel Prize in physiology and medicines in 2007 (Capecchi, 2005a). Many genome editing tools have been deployed to obtain knockouts, including nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA-guided CRISPR-Cas family, and gene targeting mediated by endogenous homology-directed repair (HDR) (Capecchi, 2005b; Miller et al, 2007; Sander et al, 2011; Wood et al, 2011; Cong et al, 2013; Knott and Doudna, 2018). Yeast exhibits high efficiency of gene targeting The mouse is another model organism traditionally deployed to study gene knockouts, in which researchers can use precise genome editing tools (Bouabe and Okkenhaug, 2013). The predominant phase in the P. patens life cycle, from the germination of spores to the fertilization of eggs, is haploid, which allows screening of knockout mutant could complete in one generation

Design of Knockout Constructs

Findings

CONCLUSIONS