Multiplex Genome-Editing Technologies for Revolutionizing Plant Biology and Crop Improvement

Abstract

Multiplex genome-editing (MGE) technologies are recently developed versatile bioengineering tools for modifying two or more specific DNA loci in a genome with high precision. These genome-editing tools have greatly increased the feasibility of introducing desired changes at multiple nucleotide levels into a target genome. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) [CRISPR/Cas] system-based MGE tools allow the simultaneous generation of direct mutations precisely at multiple loci in a gene or multiple genes. MGE is enhancing the field of plant molecular biology and providing capabilities for revolutionizing modern crop-breeding methods as it was virtually impossible to edit genomes so precisely at the single base-pair level with prior genome-editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Recently, researchers have not only started using MGE tools to advance genome-editing applications in certain plant science fields but also have attempted to decipher and answer basic questions related to plant biology. In this review, we discuss the current progress that has been made toward the development and utilization of MGE tools with an emphasis on the improvements in plant biology after the discovery of CRISPR/Cas9. Furthermore, the most recent advancements involving CRISPR/Cas applications for editing multiple loci or genes are described. Finally, insights into the strengths and importance of MGE technology in advancing crop-improvement programs are presented.

Multiplexed Genome Editing for Efficient Phenotypic Screening in Zebrafish

“…The traditional construction of multiple homozygous zebrafish mutants is time-consuming and inefficient. Multiplex genome editing technology, a reliable gene editing tool developed in recent years, has greatly enhanced the scope and efficiency of gene editing [32]. Herein, a novel strategy is proposed for multiplex genome editing that can simultaneously target five genes and rapidly obtain homozygous mutants.…”

Natural and artificial sources of genetic variation used in crop breeding: A baseline comparator for genome editing

“…Although, as knowledge of gene function and interaction improve, SDN-2 and -3 edits, along with base editing and modification of the epigenome, are set to enable designed alteration of enzymes, storage or structural proteins and transcription factors. In addition, the multiplexing of trait enhancement in these crops will become ever more routine as the market for these crops grows, and the technology becomes further refined ( Najera et al, 2019 ; Abdelrahman et al, 2021 ). Indeed, it is the ability to rapidly multiplex many changes into the same individual without the constraints of linkage drag and multiple back-crossings to a recurrent parent that make genome editing particularly advantageous.…”

Molecular and Computational Strategies to Increase the Efficiency of CRISPR-Based Techniques

“…Multiplex genome editing allows the introduction of two or more precise gene mutations in one plant generation and has the potential to significantly accelerate breeding processes ( Abdelrahman et al, 2021 ). Several multiplex Cas12a systems in plants have been developed, but the efficiency of high biallelic editing frequencies in all targeted genes were very low ( Wang et al, 2017b , 2018 ; Tang et al, 2019 ).…”