Optimized parameters for Cas9 CRISPR interference library design

作者：Smriti Srikanth, Fengyi Zheng, Laura M Drepanos, Spencer Shah, E L Kaplan, Daytan Gibson, Grace O Lynch, Allison Uebele, Ganna Reint, Sarra Merzouk, John G. Doench · 发表于：bioRxiv (Cold Spring Harbor Laboratory) · 年份：2026 · DOI：10.64898/2026.02.25.708018 · 被引用次数：2 · 研究领域：CRISPR and Genetic Engineering、Single-cell and spatial transcriptomics、Retinal Development and Disorders

ABSTRACT CRISPR interference (CRISPRi) is a powerful technology for studying loss-of-function phenotypes, enabling transient and reversible control of gene expression without the introduction of double-stranded DNA breaks. The cost of conducting large-scale CRISPR screens necessitates the selection of effective and specific sgRNAs for the design of compact libraries. While several genome-wide Cas9 CRISPRi libraries have been created, updates to transcript annotations, the generation of higher-resolution chromatin accessibility datasets and the development of newer on-target prediction models motivate an updated CRISPRi library design approach. Here, we generate large CRISPRi datasets tiling essential and nonessential genes. We compare the performance of multiple KRAB domain systems, develop an updated CRISPRi-specific on-target scoring scheme, and quantitatively characterize off-target effects associated with seed sequence patterns. We leverage these findings to design an optimized Cas9 CRISPRi library, Katsano, and validate its performance with genome-wide viability screens.