Abstract
BackgroundWhile pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have thus far not investigated the influence of cellular heterogeneity on screen results. Instead most screens are analyzed by averaging the abundance of perturbed cells from a bulk population of cells.ResultsHere we developed multi-level barcoded sgRNA libraries to trace multiple clonal Cas9 cell lines exposed to the same environment. The first level of barcoding allows monitoring growth kinetics and treatment responses of multiplexed clonal cell lines under identical conditions while the second level enables in-sample replication and tracing of sub-clonal lineages of cells expressing the same sgRNA.ConclusionUsing our approach, we illustrate how heterogeneity in growth kinetics and treatment response of clonal cell lines impairs the results of pooled genetic screens.
Highlights
While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have far not investigated the influence of cellular heterogeneity on screen results
To enable the study of screening replicates exposed to the same conditions and dissect the influence of cellular heterogeneity on the results of pooled genetic screens, we developed a multi-level barcoding strategy for pooled single guide RNA libraries
To investigate the impact of the observed heterogeneity on CRISPR screens, one resistant (CloneR) and one sensitive clone (CloneS) from either CRISPR system was used for pooled screens to identify genes involved in TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis (Fig. 1b)
Summary
While pooled loss- and gain-of-function CRISPR screening approaches have become increasingly popular to systematically investigate mammalian gene function, the large majority of them have far not investigated the influence of cellular heterogeneity on screen results. Pooled genetic screens are a powerful tool to functionally dissect genetic networks in mammalian cells and in conjunction with recently developed CRISPR/Cas systems, they permit a variety of scalable genetic perturbations, including gene knockout, knockdown or activation [1, 2]. To enable the study of screening replicates exposed to the same conditions and dissect the influence of cellular heterogeneity on the results of pooled genetic screens, we developed a multi-level barcoding strategy for pooled single guide RNA (sgRNA) libraries. The BC on the other hand, allows the analysis of sub-clonal lineages of cells expressing a certain sgRNA sequence, similar to recently described random sequence labels [10] or unique molecular identifiers [11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
