The CRISPR-Cas9 system, which uses sgRNA for targeting and the nuclease Cas9 for cleavage, has emerged as a versatile and efficient tool for genome engineering; the system has overcome the limitations of previous technologies and implements various gene editing strategies. The large-scale loss-of-function (LOF) gene scanning technology based on the CRISPR-Cas9 system can be utilized to reveal associations between the genotype and phenotype by inducing efficient and scalable gene perturbations throughout the whole genome. This technology is playing a breakthrough role in the exploration of genes associated with tumors and viruses and its expansion together with RNA sequencing (RNA-seq) can be used to assess multiplexed gene interactions as well as more complicated phenotypes. Here, we introduce LOF genetic screens based on CRISPR knockout (CRISPR ko) and its applications in synthetic lethality and virus-host interactions. We highlight the recent progress on combining this technique with single cell RNA-seq. We also compare the advantages and pitfalls of CRISPR variants, discuss the future perspectives of gene therapy and raise important considerations regarding off-target effects.

CRISPR-Cas9 系统使用 sgRNA 进行靶向，使用核酸酶 Cas9 进行切割，已成为基因组工程的多功能高效工具；该系统克服了以往技术的局限性，实现了多种基因编辑策略。基于 CRISPR-Cas9 系统的大规模功能丧失 (LOF) 基因扫描技术可用于通过在整个基因组中诱导有效且可扩展的基因扰动来揭示基因型和表型之间的关联。该技术在探索与肿瘤和病毒相关的基因方面发挥着突破性作用，其扩展与 RNA 测序 (RNA-seq) 一起可用于评估多重基因相互作用以及更复杂的表型。这里，我们介绍了基于 CRISPR 敲除 (CRISPR ko) 的 LOF 遗传筛选及其在合成致死率和病毒-宿主相互作用中的应用。我们重点介绍了将这项技术与单细胞 RNA-seq 相结合的最新进展。我们还比较了 CRISPR 变体的优点和缺陷，讨论了基因治疗的未来前景，并提出了关于脱靶效应的重要考虑因素。