College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China.
Nucleic Acids Res. 2022 Jun 24;50(11):6575-6586. doi: 10.1093/nar/gkac458.
Simultaneous targeting multiple genes is a big advantage of CRISPR (clustered regularly interspaced short palindromic repeats) genome editing but challenging to achieve in CRISPR screening. The crosstalk among genes or gene products is a common and fundamental mechanism to ensure cellular stability and functional diversity. However, the screening approach to map high-order gene combinations to the interesting phenotype is still lacking. Here, we developed a universal in-library ligation strategy and applied it to generate multiplexed CRISPR library, which could perturb four pre-designed targets in a cell. We conducted in vivo CRISPR screening for potential guide RNA (gRNA) combinations inducing anti-tumor immune responses. Simultaneously disturbing a combination of three checkpoints in CD8+ T cells was demonstrated to be more effective than disturbing Pdcd1 only for T cell activation in the tumor environment. This study developed a novel in-library ligation strategy to facilitate the multiplexed CRISPR screening, which could extend our ability to explore the combinatorial outcomes from coordinated gene behaviors.
同时靶向多个基因是 CRISPR（成簇规律间隔短回文重复序列）基因组编辑的一大优势，但在 CRISPR 筛选中很难实现。基因或基因产物之间的串扰是确保细胞稳定性和功能多样性的常见且基本的机制。然而，用于将高阶基因组合映射到感兴趣的表型的筛选方法仍然缺乏。在这里，我们开发了一种通用的文库内连接策略，并将其应用于生成可同时扰动细胞中四个预先设计的靶标的多路复用 CRISPR 文库。我们在体内进行了 CRISPR 筛选，以寻找潜在的诱导抗肿瘤免疫反应的向导 RNA（gRNA）组合。同时扰乱 CD8+T 细胞中三个检查点的组合被证明比仅扰乱 Pdcd1 更有效地激活肿瘤微环境中的 T 细胞。这项研究开发了一种新的文库内连接策略，以促进多路复用 CRISPR 筛选，这将扩展我们探索协调基因行为的组合结果的能力。
