CRISPR/Cas9 Genome Editing Technology: A Valuable Tool for Understanding Plant Cell Wall Biosynthesis and Function

Abstract

For the past 5 years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has appeared in the molecular biology research spotlight. As a game-changing player in genome editing, CRISPR/Cas9 technology has revolutionized animal research, including medical research and human gene therapy as well as plant science research, particularly for crop improvement. One of the most common applications of CRISPR/Cas9 is to generate genetic knock-out mutants. Recently, several multiplex genome editing approaches utilizing CRISPR/Cas9 were developed and applied in various aspects of plant research. Here we summarize these approaches as they relate to plants, particularly with respect to understanding the biosynthesis and function of the plant cell wall. The plant cell wall is a polysaccharide-rich cell structure that is vital to plant cell formation, growth, and development. Humans are heavily dependent on the byproducts of the plant cell wall such as shelter, food, clothes, and fuel. Genes involved in the assembly of the plant cell wall are often highly redundant. To identify these redundant genes, higher-order knock-out mutants need to be generated, which is conventionally done by genetic crossing. Compared with genetic crossing, CRISPR/Cas9 multi-gene targeting can greatly shorten the process of higher-order mutant generation and screening, which is especially useful to characterize cell wall related genes in plant species that require longer growth time. Moreover, CRISPR/Cas9 makes it possible to knock out genes when null T-DNA mutants are not available or are genetically linked. Because of these advantages, CRISPR/Cas9 is becoming an ideal and indispensable tool to perform functional studies in plant cell wall research. In this review, we provide perspectives on how to design CRISPR/Cas9 to achieve efficient gene editing and multi-gene targeting in plants. We also discuss the recent development of the virus-based CRISPR/Cas9 system and the application of CRISPR/Cas9 to knock in genes. Lastly, we summarized current progress on using CRISPR/Cas9 for the characterization of plant cell wall-related genes.

CRISPR-Cas9 multiplex genome editing of the hydroxyproline-O-galactosyltransferase gene family alters arabinogalactan-protein glycosylation and function in Arabidopsis

“…Consequently, the GTs responsible for adding the sugars to AGPs are of particular interest. Studying these GTs can be particularly challenging for several reasons, including the finding that these GTs exist in gene families and have overlapping and redundant activities, which makes it difficult to examine their effects on AGP structure and function [26,27]. showed a delayed germination phenotype.…”

“…Yariv treated Arabidopsis roots exhibit a short, swollen root phenotype, which is caused by an inhibition of cell proliferation and cell elongation [23]. Several other AGP-related GTs or AGP mutants such as reb1-1, ray1, fut4/fut6, glcat14a-1, fla1, fla4/sos5 and agp30, also display abnormal root phenotypes under normal or salt-stress conditions in Arabidopsis [12][13][14][26][27][28][29]. In addition, AGPs antibodies such as JIM8, JIM13, and LM2 localize AGPs in primary roots, root caps, and root hairs [30][31][32].…”

Microbial Cell Factories for Green Production of Vitamins

“…In general, the development of synthetic biotechnology provides new opportunities for the construction of vitamin cell factories. First, high-throughput screening of high-yield strains, the CRISPR/Cas9 genome editing technology, and automatic gene assembly technology provide important technical means for the mining and genetic modification of chassis cells (Chang et al, 2019;Zeng W. et al, 2020;Zhang and Showalter, 2020). Further, the output of vitamin products in different dimensions will be increased by transforming the complex and multi-enzyme pathways required for the production of vitamins, establishing microbial flora with controllable functions and stability, and application of some advanced engineering technology, such as the cold-shock-triggered temperature control system, dynamic control of gene expression systems, different types of biosensors, cell-free systems and computer-aided design, etc.…”

Plant Cell Wall as a Key Player During Resistant and Susceptible Plant-Virus Interactions

“…In particular, the CRISPR/ Cas9 system may provide an insight into the genetic control of the structure and functions of plant cell wall (Whitehead et al, 2018;Cao et al, 2020;McCarty et al, 2020). At present, these methods are generally used in studies focusing on cell wall synthesis pathways (including enzymes) and analyzing the exact functions of cell wall elements (Zhang and Showalter, 2020) for the improvement of crop products Wang D. et al, 2019). However, Makarova et al (2018) indicated the application of the CRISPR/Cas9 system for generating plant resistance (not precisely associated with cell wall) against various pathogens.…”