Minimal genome-wide human CRISPR-Cas9 library

Abstract

CRISPR guide RNA libraries have been iteratively improved to provide increasingly efficient reagents, although their large size is a barrier for many applications. We design an optimised minimal genome-wide human CRISPR-Cas9 library (MinLibCas9) by mining existing large-scale gene loss-of-function datasets, resulting in a greater than 42% reduction in size compared to other CRISPR-Cas9 libraries while preserving assay sensitivity and specificity. MinLibCas9 provides backward compatibility with existing datasets, increases the dynamic range of CRISPR-Cas9 screens and extends their application to complex models and assays.

Keywords: CRISPR-Cas9; Genome-wide; KS score; Minimal library; Organoid.

Fig. 1

Genome-wide human CRISPR-Cas9 sgRNA libraries. a Number of sgRNAs in each CRISPR-Cas9 library since the first reported genome-wide screens, excluding Wang et al. [3] which targets 7114 genes. b Area under the recall curve of sgRNAs targeting known essential (n = 1469) and non-essential (n = 3251) genes, and non-targeting guides (n = 997). Recall curves were calculated for each replicate of Project Score [13] (n = 663) and represented by the cumulative distribution of each sgRNA group across all sgRNAs sorted by ascending fold-changes. Box-and-whisker plots show 1.5× interquartile ranges and 5–95th percentiles, centres indicate medians. c Fold-change distribution, based on Project Score data-set of the different sgRNAs groups. Diagram depicting how the KS scores vary across the CRISPR-screens fold-change range. KS scores are calculated by testing if the distribution of each sgRNA across cell lines is drawn from that of the non-targeting sgRNAs using a two-sided Kolmogorov-Smirnov distribution

Fig. 2

Benchmark of MinLibCas9 library. a Standardised area under the receiver operating characteristic curve for 245 cell lines at 20% FDR for the essential genes calculated using the minimal and original full library. b Average Precision (AP) scores to classify significant gene dependencies identified at 1% FDR in Project Score library using gene fold-changes from MinLibCas9. Recall-Precision curves for all cell lines are represented in the inset and cell lines with the lowest and highest AP score are highlighted. c, CRISPR-Cas9 screen performed on HT-29 cancer cells using MinLibCas9 library. d Correlation between gene-level fold-changes obtained with the two libraries. e Recall of essential genes across all replicates for HT-29 performed with MinLibCas9 and Project Score libraries