ABSTRACT
CRISPR/Cas-based editing has advanced dramatically recently, offering a multitude of progressive approaches, capable of inducing almost any type of desired modification in the genome of various organisms. One of such advancements is multiplex editing, since CRISPR/Cas system is the most favourable platform for multiplexing, making it a technique of choice. In some cases, the selected targets may be in the same gene and in others, they may be in different genes, the latter being feasible as the sgRNAs targeting the genes can be independently expressed in cassettes with individual promoters or with a single promoter as is the case in polycistronic expression system. The size of the sgRNA- expressing construct is a factor limiting the multiplex editing efficiency and the targeting capability of the CRISPR/Cas technique. Various methods have been developed aiming to mitigate this issue, including tRNA, Csy4, miRNA, and ribozyme-based in vivo sgRNA processing systems. Multiplexing has even been utilized in advanced CRISPR/Cas platforms such as Cas/gRNA ribonucleoprotein (RNPs)-based systems, precise base editing, promoter bashing, and prime editing. Here, we present advanced approaches for enhancing CRISPR/Cas applications and multiplexing efficiency in plants, potentially widening the scope of crop improvement programs. In the present chapter, different approaches for multiplex genome editing and their potential applications, as well as challenges to their efficient exploration are discussed.
