CRISPR-Cas9–based screening with single-guide RNA (sgRNA) libraries has emerged as a revolutionary tool for comprehensive analysis of genetic elements. However, genome-scale sgRNA libraries are currently available only in a few model organisms. The traditional approach is to synthesize thousands to tens of thousands of sgRNAs, which is laborious and expensive. We have developed a simple method, RELATe (restriction/ligation coupled with Agrobacterium-mediated transformation), to generate sgRNA libraries from 10 μg of genomic DNA, targeting over 98% of the protein-coding genes in the human fungal pathogen Cryptococcus neoformans. Functional screens identified 142 potential C. neoformans genes contributing to blood-brain barrier penetration. We selected two cryptococcal genes, SFP1 and WDR1, for a proof-of-concept demonstration that RELATe-identified genes are relevant to C. neoformans central nervous system infection. Our RELATe method can be used in many other fungal species and is powerful and cost-effective for genome-wide high-throughput screening for elucidating functional genomics.

基于 CRISPR-Cas9 的单向导 RNA (sgRNA) 文库筛选已成为综合分析遗传元件的革命性工具。然而，基因组规模的 sgRNA 文库目前仅在少数模式生物中可用。传统的方法是合成数千至数万个sgRNA，既费力又昂贵。我们开发了一种简单的方法 RELATe（限制/连接加上农杆菌介导的转化），从 10 μg 基因组 DNA 生成 sgRNA 文库，靶向人类真菌病原体新型隐球菌中超过 98% 的蛋白质编码基因。功能筛选鉴定出 142 个潜在的新型隐球菌基因，有助于穿透血脑屏障。我们选择了两个隐球菌基因SFP1和WDR1来进行概念验证，证明 RELATe 鉴定的基因与新型隐球菌中枢神经系统感染相关。我们的 RELATe 方法可用于许多其他真菌物种，对于阐明功能基因组学的全基因组高通量筛选来说功能强大且经济高效。