Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library
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METHODOLOGY ARTICLE
Open Access
Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library Luisa Henkel†, Benedikt Rauscher†, Barbara Schmitt, Jan Winter and Michael Boutros*
Abstract Background: In recent years, large-scale genetic screens using the CRISPR/Cas9 system have emerged as scalable approaches able to interrogate gene function with unprecedented efficiency and specificity in various biological contexts. By this means, functional dependencies on both the protein-coding and noncoding genome of numerous cell types in different organisms have been interrogated. However, screening designs vary greatly and criteria for optimal experimental implementation and library composition are still emerging. Given their broad utility in functionally annotating genomes, the application and interpretation of genome-scale CRISPR screens would greatly benefit from consistent and optimal design criteria. Results: We report advantages of conducting viability screens in selected Cas9 single-cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify singleguide (sg) RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a novel genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. Conclusion: We show how empirically designed libraries in combination with an optimized experimental design increase the dynamic range in gene essentiality screens at reduced library coverage. Keywords: CRISPR/Cas9, Genetic screens, sgRNA design, Gene essentiality, Functional genomics
Background Over the past decades, genetic screens have been used extensively to interrogate gene function in an unbiased manner [1–3]. In recent years, CRISPR/Cas9 [4, 5] has emerged as a scalable method to introduce targeted gene knockouts with unprecedented efficiency and specificity. Genetic screens with the CRISPR/Cas9 system are now applied to probe the protein-coding and noncoding genomes of hundreds of cell types in different organisms [6–11]. These experiments have led to new insights into many biological processes. Progress has been made * Correspondence: [email protected] † Luisa Henkel and Benedikt Rauscher contributed equally to this work. German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics and Heidelberg University, BioQuant and Medical Faculty Mannheim, D-69120 Heidelberg, Germany
especially in the field of cancer genetics, where genomewide CRISPR screens have resulted in gene essentiality maps for hundreds of tumor cell lines [12, 13]. Despite these advances, currently no generally accepted design principles for sgRNA libraries [14] and large-scale CRISPR screens exist and published experiments vary substantially in design and performance. This is particularly important as we reason that screens conducted with high CRISPR editing efficiency based on high Cas9 and sgRNA functionality allow screens at reduced coverage, which s
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