CRISPR-UMI: single-cell lineage tracing of pooled CRISPR–Cas9 screens

Abstract

Pooled CRISPR screens are a powerful tool for assessments of gene function. However, conventional analysis is based exclusively on the relative abundance of integrated single guide RNAs (sgRNAs) between populations, which does not discern distinct phenotypes and editing outcomes generated by identical sgRNAs. Here we present CRISPR-UMI, a single-cell lineage-tracing methodology for pooled screening to account for cell heterogeneity. We generated complex sgRNA libraries with unique molecular identifiers (UMIs) that allowed for screening of clonally expanded, individually tagged cells. A proof-of-principle CRISPR-UMI negative-selection screen provided increased sensitivity and robustness compared with conventional analysis by accounting for underlying cellular and editing-outcome heterogeneity and detection of outlier clones. Furthermore, a CRISPR-UMI positive-selection screen uncovered new roadblocks in reprogramming mouse embryonic fibroblasts as pluripotent stem cells, distinguishing reprogramming frequency and speed (i.e., effect size and probability). CRISPR-UMI boosts the predictive power, sensitivity, and information content of pooled CRISPR screens.

Microenvironmental regulation of cancer cell metabolism: implications for experimental design and translational studies

“…New theoretical and computational tools to measure intercell metabolite sharing between different cell types using stable isotope tracing (Gebreselassie and Antoniewicz, 2015; Ghosh et al, 2014; Rossi et al, 2017; Ruhl et al, 2011; Shaikh et al, 2008) might also facilitate the study of metabolic commensalism in tumors. New developments in CRISPR-based genetic screening approaches could make the functional screening of metabolic pathway requirements possible in mouse cancer models (Michlits et al, 2017; Chow and Chen, 2018; Schmierer et al, 2017). Furthermore, revisiting old techniques with new analytical technologies could also provide new insight into tumor metabolism.…”

Minimized double guide RNA libraries enable scale-limited CRISPR/Cas9 screens

“…This enables customizable experimental design, where the same gRNA sub-pools could be combined depending on the desired tests. In principle, we could further introduce a multi-gRNA expression construct (Liu et al 2019) , include unique molecular identifiers (Michlits et al 2017;Schmierer et al 2017) , and insert additional expression constructs (Martella et al 2017) to combine the benefits of the different approaches. Care is needed to ensure equal efficacy and representation of all products in these highly multiplexed settings.…”

A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

“…Rather than sequencing guides directly, we used arbitrary nucleotide barcodes embedded in the guide RNA expression plasmid. One major advantage of sequencing these barcodes is that each guide can be linked to a few different barcodes, providing replicate measurements of its effect within a single experiment (Michlits et al, 2017;Schmierer et al, 2017). In contrast, direct guide sequencing cannot distinguish between independently transformed lineages within a single experiment.…”