CRISPR-UMI Step by Step: A protocol for robust CRISPR screening

Abstract

CRISPR-UMI extends the existing repertoire of CRSIPR-screening methods. It circumvents cell heterogeneity, a consequence of Cas9 genome editing, by scoring single cell derived clones individually. The strength of this new CRISPR screening method is its robustness towards clonal heterogeneity and clonal outliers and is therefore expected to be most useful in challenging biological screens with strong bottlenecks and clonal effects such as organoid or in-vivo screens. This step-by-step protocol is an addition to the publication CRISPR-UMI: Single cell lineage tracing of pooled CRISPR/Cas9 screens doi: 10.1038/nmeth.4466. It contains a detailed description for pooled CRISPR screening using CRISPR-UMI. It especially highlights the steps which are critical and unique to the use of CRISPR-UMI. Those critical steps are library preparation at very high complexity of up to 100million unique plasmids, and data analysis were unique guide-UMI pairs are evaluated separately.