Applications of multiplex genome editing in higher plants

• Published in: Current opinion in biotechnology Vol. 59; pp. 93 - 102
• Main Authors: Armario Najera, Victoria, Twyman, Richard M, Christou, Paul, Zhu, Changfu
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-10-2019
• Subjects: Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas Systems; Gene Editing; Genome, Plant; Plants; Plants, Genetically Modified; RNA, Guide, CRISPR-Cas Systems
• ISSN: 0958-1669; 1879-0429
• DOI: 10.1016/j.copbio.2019.02.015

Multiplex genome editing in plants is typically carried out using the CRISPR/Cas9 system because it is more amenable to multiplexing than any other platform. A single gRNA can be used to target multiple alleles of the same gene (A1, A2) or homeoalleles from different ancestral genomes in a polyploid plant (A1, A2, B1, B2). A single gRNA can also be used to target multiple genes (X, Y, Z) if they contain a conserved region (C). Multiple gRNAs can be used to target unrelated sites in the same gene or in conserved sequences such as homeoalleles or members of the same gene family. Multiple gRNAs can also be used to target completely unrelated genes (D, E, F).
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•Multiplex genome editing can target multiple similar and/or dissimilar sequences.•Multiple targets may be inside the same gene, (homeo)alleles/paralogs, or different genes.•The highest number of multiple targeting events reported thus far in plants is 107.•The coding region is usually targeted but promoter targeting can also generate new alleles.•Multiplex genome editing applications include metabolic engineering and molecular farming.
Multiplex genome editing involves the simultaneous targeting of multiple related or unrelated targets. The latter is most straightforward using the CRISPR/Cas9 system because multiple gRNAs can be delivered either as independent expression cassettes with their own promoters or as polycistronic transcripts processed into mature gRNAs by endogenous or introduced nucleases. Multiplex genome editing in plants initially focused on input traits such as herbicide resistance, but has recently expanded to include hormone biosynthesis and perception, metabolic engineering, plant development and molecular farming, with more than 100 simultaneous targeting events reported. Usually the coding region is targeted but recent examples also include promoter modifications to generate mutants with varying levels of gene expression.