Cell genomicsAug 28, 2026

Best settings for designing CRISPR-Cas9 gene interference libraries

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Aug 29, 2026

Abstract

An optimized CRISPRi-Cas9 library, Katsano, was developed and validated with genome-wide viability screens.

CRISPR interference (CRISPRi) allows for reversible control of gene expression without double-stranded DNA breaks.
The study generated large CRISPRi datasets that cover both essential and nonessential genes.
Multiple KRAB domain systems were compared to enhance the effectiveness of CRISPRi.
An updated scoring scheme for on-target prediction was developed specifically for CRISPRi applications.
Quantitative analysis of off-target effects revealed associations with specific seed-sequence patterns.

Simplified

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 single-guide RNAs for the design of compact libraries. While several genome-wide CRISPRi-Cas9 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 CRISPRi-Cas9 library, Katsano, and validate its performance with genome-wide viability screens.

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Best settings for designing CRISPR-Cas9 gene interference libraries

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