Targeted Gene Knockouts by Protoplast Transformation in the Moss Physcomitrella patens

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.

Identification and Characterization of an Antifungal Gene Mt1 from Bacillus subtilis by Affecting Amino Acid Metabolism in Fusarium graminearum

“…The library preparation for whole-genome sequencing (WGS) using next-generation sequencing (NGS) begins with DNA fragmentation, and sonication is the most commonly used approach due to its ease and reliability [15,16]. In recent years, PEG-mediated protoplast transformation and homologous recombination methods for knocking out and knocking in target genes have become widely popular, and researchers using both can study functional genes, metabolic regulatory networks, and genetic engineering in plants, animals, and microorganisms [17][18][19][20][21]. Gene knock-in allows researchers to study the function of specific genes in fungi as well as engineer fungi for various biotechnological applications [22][23][24].…”