In genome editing applications, multiplexing may refer to targeting two or more loci by a common/shared target site, or to using multiple programmable nucleases to target several independent sites. Multiplex genome-editing (MGE) technologies are developing versatile bioengineering tools for modifying two or more specific DNA loci in a genome with high precision. The great advantage of this strategy over traditional complex hybridisation programs is that we can target multiple sites simultaneously and create new lines carrying several mutations in one generation. MGE can be used to target multiple genes, multiple sites in the same gene, multiple gene variants or members of gene families, to introduce larger mutations, and to regulate gene expression at the genetic and epigenetic levels1 . MGE tools are designed to target specific genomic regions and to generate specific mutations or variations with much more precision. Among genome-editing technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), researchers have preferred CRISPR-Cas9 system due to its simplicity for designing multiple target genetic loci. Nonetheless, an impressive 107/109 genes encoding caffeic acid O-methyltransferase has been edited using a single TALEN pair in sugarcane. CRISPR/Cas9 system provide much more flexibility for the editing of targets because it is less expensive to provide multiple gRNAs. There are two broad strategies to introduce multiple gRNAs: The gRNAs can be provided as individual expression cassettes or they can be expressed as a polycistronic transcript with post transcriptional processing 1 .

MGE is enhancing the field of plant molecular biology and providing capabilities for revolutionizing modern crop-breeding methods. In Tomato, fruit colour is an important horticultural trait, which greatly affects consumer preferences. A rapid breeding strategy has been proposed to generate different coloured fruits from red-fruited materials by CRISPR/Cas9-mediated multiplex gene editing of three fruit colour-related genes (PSY1, MYB12, and SGR1). Using this strategy, the red-fruited cultivar 'Ailsa Craig' has been engineered to a series of tomato genotypes with different fruit colors2 .

A study proposed a new breeding strategy in rice, where they simultaneously targeted three yield-related QTLs, OsGS3, OsGW2 and OsGn1a in contrary to the traditional breeding approach which require multiple rounds of crossing and selection. Through this approach, seven combinations of single, double and triple mutants are generated for the target genes in elite backgrounds which revealed significant increase in the yield performance3. Applications of MGE have been efficiently utilized to enhance crop yield, quality, and stress resistance and its implementation is required for facilitating new discoveries in the field of agriculture.

References

1ABDELRAHMAN, M., WEI, Z., ROHILA, J. S. AND ZHAO, K., 2021, Multiplex genome-editing technologies for revolutionizing plant biology and crop improvement. Front. Plant Sci., 12.

2YANG, T., ALI, M., LIN, L., LI, P., HE, H., ZHU, Q., SUN, C., WU, N., ZHANG, X., HUANG, T., LI, C. B., LI, C. AND DENG, L., 2023, Recolouring tomato fruit by CRISPR/Cas9-mediated multiplex gene editing. Hortic. Res., 10(1).

3ZHOU, J., XIN, X., HE, Y., CHEN, H., LI, Q., TANG, X., ZHONG, Z., DENG, K., ZHENG, X., AKHER, S. A., CAI, G., QI, Y. AND ZHANG, Y., 2019, Multiplex QTL editing of grain-related genes improves yield in elite rice varieties. Plant Cell Rep., 38(2).