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

编号 040035605

推送时间 20220815

研究领域 森林培育 

年份 2022 

类型 期刊 

语种 中文

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

来源期刊 Frontiers in plant science

期 第356期

发表时间 20220620

关键词 combinatorial gene knockout libraries;  crop breeding;  experimental design;  multiplex CRISPR/Cas screens;  plant genetic studies.; 

摘要       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.

服务人员 孙小满

服务院士 尹伟伦

PDF文件 浏览全文