College of Life Sciences, Zhejiang University, Hangzhou, 310058, China.
School of Life Sciences, Westlake University, 600 Dunyu Road, Hangzhou, 310030, Zhejiang, China.
Sci Rep. 2023 Jul 18;13(1):11569. doi: 10.1038/s41598-023-38810-6.
Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screening is revolting the genetic analysis of a cellular or molecular phenotype in question but is challenged by the large size of single-guide RNA (sgRNA) library. Here we designed a minimal genome-wide human sgRNA library, H-mLib, which is composed of 21,159 sgRNA pairs assembled based on a dedicated selection strategy from all potential SpCas9/sgRNAs in the human genome. These sgRNA pairs were cloned into a dual-gRNA vector each targeting one gene, resulting in a compact library size nearly identical to the number of human protein-coding genes. The performance of the H-mLib was benchmarked to other CRISPR libraries in a proliferation screening conducted in K562 cells. We also identified groups of core essential genes and cell-type specific essential genes by comparing the screening results from the K562 and Jurkat cells. Together, the H-mLib exemplified high specificity and sensitivity in identifying essential genes while containing minimal library complexity, emphasizing its advantages and applications in CRISPR screening with limited cell numbers.
基于基因组广泛聚集的规律间隔短回文重复序列 (CRISPR) 的敲除筛选正在颠覆人们对细胞或分子表型的遗传分析，但受到单指导 RNA (sgRNA) 文库规模大的挑战。在这里，我们设计了一个最小的人类基因组广泛 sgRNA 文库 H-mLib，它由 21159 对 sgRNA 组成，这些 sgRNA 是根据人类基因组中所有潜在 SpCas9/sgRNA 进行专门选择策略组装而成的。这些 sgRNA 对被克隆到一个双 sgRNA 载体中，每个载体靶向一个基因，导致文库大小紧凑，几乎与人类编码蛋白基因的数量相同。在 K562 细胞中进行的增殖筛选中，我们将 H-mLib 的性能与其他 CRISPR 文库进行了基准测试。我们还通过比较 K562 和 Jurkat 细胞的筛选结果，鉴定了核心必需基因和细胞类型特异性必需基因的群组。总之，H-mLib 在鉴定必需基因方面表现出了高特异性和灵敏度，同时文库复杂性最小，强调了其在有限细胞数量的 CRISPR 筛选中的优势和应用。
