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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have