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Covering the Combinatorial Design Space of Multiplex CRISPR/Cas Experiments in Plants.

ABSTRACT: Over the past years, CRISPR/Cas-mediated genome editing has revolutionized plant genetic studies and crop breeding. Specifically, due to its ability to simultaneously target multiple genes, the multiplex CRISPR/Cas system has emerged as a powerful technology for functional analysis of genetic pathways. As such, it holds great potential for application in plant systems to discover genetic interactions and to improve polygenic agronomic traits in crop breeding. However, optimal experimental design regarding coverage of the combinatorial design space in multiplex CRISPR/Cas screens remains largely unexplored. To contribute to well-informed experimental design of such screens in plants, we first establish a representation of the design space at different stages of a multiplex CRISPR/Cas experiment. We provide two independent computational approaches yielding insights into the plant library size guaranteeing full coverage of all relevant multiplex combinations of gene knockouts in a specific multiplex CRISPR/Cas screen. These frameworks take into account several design parameters (e.g., the number of target genes, the number of gRNAs designed per gene, and the number of elements in the combinatorial array) and efficiencies at subsequent stages of a multiplex CRISPR/Cas experiment (e.g., the distribution of gRNA/Cas delivery, gRNA-specific mutation efficiency, and knockout efficiency). With this work, we intend to raise awareness about the limitations regarding the number of target genes and order of genetic interaction that can be realistically analyzed in multiplex CRISPR/Cas experiments with a given number of plants. Finally, we establish guidelines for designing multiplex CRISPR/Cas experiments with an optimal coverage of the combinatorial design space at minimal plant library size.

SUBMITTER: Van Huffel K

PROVIDER: S-EPMC9251496 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, the design and assembly of multiplex constructs comprising tandemly arrayed guide RNAs (gRNAs) requires scarless cloning and is still troublesome due to the presence of repetitive sequences, thus hampering a more widespread use. Here we present a comprehensive extension of the software-assisted cloning platform GoldenBraid (GB), in which, on top of its multigene cloning software, we integrate new tools for the Type IIS-based easy and rapid assembly of up to six tandemly-arrayed gRNAs with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the crRNA unspaced approaches, respectively. As stress tests for the new tools, we assembled and used for Agrobacterium-mediated stable transformation a 17 Cas9-gRNAs construct targeting a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in Nicotiana tabacum. The 14 selected genes are targets of miR156, thus potentially playing an important role in juvenile-to-adult and vegetative-to-reproductive phase transitions. With the 17 gRNAs construct we generated a collection of Cas9-free SPL edited T1 plants harboring up to 9 biallelic mutations and showing leaf juvenility and more branching. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR/Cas activators and repressors using single and multiplexing gRNAs was validated using a Luciferase reporter with the Solanum lycopersicum Mtb promoter or the Agrobacterium tumefaciens nopaline synthase promoter in transient expression in Nicotiana benthamiana. With the incorporation of the new web-based tools and the accompanying collection of DNA parts, the GB4.0 genome edition turns an all-in-one open platform for plant genome engineering.

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Covering the Combinatorial Design Space of Multiplex CRISPR/Cas Experiments in Plants.

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